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Whole-brain irradiation differentially modifies neurotransmitters levels and receptors in the hypothalamus and the prefrontal cortex

Whole-brain irradiation differentially modifies neurotransmitters levels and receptors in the... Background: Whole‑brain radiotherapy is a primary treatment for brain tumors and brain metastasis, but it also induces long‑term undesired effects. Since cognitive impairment can occur, research on the etiology of secondary effects has focused on the hippocampus. Often overlooked, the hypothalamus controls critical homeostatic functions, some of which are also susceptible after whole‑brain radiotherapy. Therefore, using whole ‑brain irradiation ( WBI) in a rat model, we measured neurotransmitters and receptors in the hypothalamus. The prefrontal cortex and brainstem were also analyzed since they are highly connected to the hypothalamus and its regulatory processes. Methods: Male Wistar rats were exposed to WBI with 11 Gy (Biologically Eec ff tive Dose = 72 Gy). After 1 month, we evaluated changes in gamma‑aminobutyric acid (GABA), glycine, taurine, aspartate, glutamate, and glutamine in the hypothalamus, prefrontal cortex, and brainstem according to an HPLC method. Ratios of Glutamate/GABA and Glutamine/Glutamate were calculated. Through Western Blott analysis, we measured the expression of GABAa and GABAb receptors, and NR1 and NR2A subunits of NMDA receptors. Changes were analyzed comparing results with sham controls using the non‑parametric Mann–Whitney U test (p < 0.05). Results: WBI with 11 Gy induced significantly lower levels of GABA, glycine, taurine, aspartate, and GABAa receptor in the hypothalamus. Also, in the hypothalamus, a higher Glutamate/GABA ratio was found after irradiation. In the prefrontal cortex, WBI induced significant increases of glutamine and glutamate, Glutamine/Glutamate ratio, and increased expression of both GABAa receptor and NMDA receptor NR1 subunit. The brainstem showed no statistically significant changes after irradiation. Conclusion: Our findings confirm that WBI can affect rat brain regions differently and opens new avenues for study. After 1 month, WBI decreases inhibitory neurotransmitters and receptors in the hypothalamus and, conversely, increases excitatory neurotransmitters and receptors in the prefrontal cortex. Increments in Glutamate/GABA in the hypothalamus and Glutamine/Glutamate in the frontal cortex indicate a neurochemical imbalance. Found changes could be related to several reported radiotherapy secondary effects, suggesting new prospects for therapeutic targets. Keywords: Whole brain irradiation, Amino acids, Neurotransmitters, Prefrontal cortex, Hypothalamus, GABAa, GABAb, NR1, NR2A Background *Correspondence: paolabaze@gmail.com Whole-brain radiotherapy is a primary medical treat- Laboratory of Medical Physics, National Institute of Neurology ment for some types of brain cancer, especially for brain and Neurosurgery, INNN, Insurgentes Sur 3877, Col. La Fama, C.P. 14269 Mexico City, Mexico metastasis. Although it improves patient’s survival, it is Full list of author information is available at the end of the article © The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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According to the time of appearance, mune stress response [18, 19]. these effects have been classified as acute (hours to days), The data presented here demonstrate that WBI pro - early-delayed (1–6 months), and late-delayed (more than duces early delayed effects in the expression of recep - 6  months after) [1]. It is currently well established that tors and neurotransmitters levels at the hypothalamus whole-brain irradiation (WBI) at therapeutic doses leads and prefrontal cortex. Its possible implications for future to an increased risk for late-delayed cognitive impair- research are discussed. ments [2]. The hippocampus is the main structure asso - ciated with memory and cognition, and thus the most Methods studied after brain irradiation. Authors have proposed Animals that the ablation of hippocampal neurogenesis plays a Twenty-four male Wistar rats (270–300  g) were used. crucial role in cognitive impairment after radiation [3, Animals were housed in a room with controlled tem- 4]. It has also been suggested that the cellular mecha- perature (22 ± 2  °C), light–dark cycle (12:12), and nism underlying cognitive deficit involves alterations in ad  libitum access to water and food. All animals were receptors related to synaptic plasticity. Therefore, using handled according to Mexican Official Norms for the in vitro and in vivo models, it has been shown that irradi- production, care, and use of laboratory animals (NOM- ation changes the expression of excitatory and inhibitory 062-Z00-1999). Additionally, the Guide for the Care and receptors and neurotransmitters in the hippocampus Use of Laboratory Animals (NIH Guide) was revised and [5–9]. used as guidelines. WBI induces a complex initial cascade of neurochemi- cal processes, which can trigger the appearance of early- Whole‑brain irradiation delayed effects often considered transient and clinically The irradiation was performed using a micro-multileaf overlooked. Early-delayed effects include Radiation Som - collimator coupled to a linear accelerator for head treat- nolence Syndrome (RSS) characterized by increased sleep ments (Novalis Varian, 6 MV), as previously reported during the day, fatigue, decreased appetite, and weight [20]. A prescription dose of 11 Gy was chosen since it cor- loss [10]. Indeed, fatigue, loss of appetite, and weakness responds to a BED (Biologically effective dose) of 72  Gy are the most frequent symptoms reported to worsen in similar to the one used clinically (10 Fx, 3 Gy α/β = 2 Gy). patients after WBI [11]. Many of these symptoms are All animals were deeply anesthetized by administering closely related to alterations of the hypothalamic func- a mixture of ketamine (100  mg/kg) as a sedative agent, tion. Moreover, it has been observed that endocrine dis- and xylazine (10  mg/kg) as a muscle relaxant. Once the ruption of the hypothalamic–pituitary–adrenal (HPA) animals showed sensory stimulation response inhibition, axis can frequently appear after WBI [12]. The hypo - they were immobilized in a custom device and fixed to thalamus is a region that controls many critical homeo- the treatment table. A single dose of the drugs was suf- static functions, including those that are perturbed after ficient to carry out the WBI. The dynamic arcs tech - whole-brain radiotherapy [12, 13]. Therefore, it is rel - nique was employed at a dose rate of 500  UM/min. As evant to analyze hypothalamic neurochemical changes with patients, homogenous coverage was achieved all after WBI. Consequently, in this work, we measured over the brain (RTOG homogeneity index HI = 1.3) while excitatory and inhibitory neurotransmitters levels in the brain surrounding structures were protected. Sham ani- hypothalamus. Additionally, we measured the expression mals were mounted but received no dose. We used rat of gamma-aminobutyric acid (GABA) receptors (GABAa CT images and the software iPlan (BrainLab Germany) and GABAb) and NMDA receptor subunits NR1 and for the treatment planning, and the dose verification was NR2A since they have previously shown variations after performed using Monte Carlo techniques [20]. different irradiation doses in other brain structures [5, 6, 14]. Moreover, we calculated Glutamate/GABA and Glu- Brain amino acids analysis tamine/Glutamate ratios as they have been used as mark- Rats were killed by decapitation 1 month after the whole ers of neurochemical brain balance [15, 16]. brain irradiation or sham manipulation. All the ani- The brain response to radiation fluctuates according mals were sacrificed at the light phase between 9:00 and to the analyzed region. Thus, Todorovic et  al. [17] dem - 10:00  a.m. to avoid circadian variations. Brain regions onstrated that the antioxidant response after radiation is dissection was based on previously published protocols lower in the hippocampus than the cerebral cortex. For [21, 22]. Briefly, after decapitation, we extracted the brain this reason, we also analyze regions such as the prefron- by opening the skull through the midline. To obtain the tal cortex and brainstem. Both structures are highly con- prefrontal cortex, we first separate the olfactory bulb. nected to the hypothalamus and are considered critical Later, with the help of a rat brain slicer matrix, we made F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 3 of 13 a 1-mm coronal cut to discard mainly the motor cor- monoclonal antibody (1:1000, sc-376282, Santa Cruz, tex, then we obtained another 1-mm coronal cut. In this USA), anti-NR1 monoclonal mouse antibody (1:200, slice, we took the genus corpus callosum and the cap- BML-SA493-0015, Enzo, USA) or anti-NR2A monoclo- sula externa as a reference to delimitate and separate the nal mouse antibody (1:200, sc-31540, Santa Cruz, USA) striatum and thus get the prefrontal cortex sample. After diluted in TBST containing 5% nonfat dry milk. After that, we placed the brain ventral side up and located the washing with TBST, membranes were incubated for 1  h optical chiasm and the midbrain in the anterior and pos- at room temperature with anti-mouse IgG HRP conju- terior parts. With the help of a spatula, we punctured gated (1:10000, SAB3701105-2, Sigma Aldrich, USA). around such anatomical structures, and thus we obtained Protein bands were observed by chemiluminescence the hypothalamus. Finally, the brainstem was dissected using Luminata Forte (Millipore, USA) and an imaging after the elimination of the cerebellum and the inferior system Fusion Solo S (Vilber, France). After detection, and superior colliculus. The brainstem portion included we submerged membranes in stripping buffer washed the medulla oblongata and pons. The brain regions were with TBST, blocked with TBST containing 5% nonfat quickly dissected using the illustrations and coordinates dry milk, and incubated with anti-α Tubulin monoclonal shown in Paxinos and Watson Atlas [23]. The samples mouse antibody (1:1000, sc23948, Santa Cruz, USA) as were stored at − 70  °C until later analysis. The aspartate protein loading reference. (Asp), GABA, glutamate (Glu), glutamine (Gln), gly- cine (Gly), and taurine (Tau) contents were determined Statistical analysis according to a method previously reported [24]. Briefly, We compared sham manipulation data with irradiated tissue was homogenized and centrifuged at 4000×g animals running the two independent samples non-para- for 10  min at 4  °C, and the supernatants were kept at metric Mann–Whitney U test in SPSS (v 20 IBM). Differ - − 70  °C until assayed. The amino acid content was ana - ences were considered significant if p ≤ 0.05. lyzed using a high-performance liquid chromatography (HPLC) system Agilent 1100 series (Agilent Technolo- gies) equipped with a fluorescence detector and Adsor - Results bosphere ortho-phthalaldehyde (OPA) column (Alltech). In the present study, Wistar rats were treated with a The mobile phase consisted of a 50-mM sodium acetate WBI protocol calculated to guarantee adjacent tissue buffer (pH 5.9) solution containing 1.5% tetrahydro - protection, especially the mucosal tract. Consequently, furan and HPLC-grade methanol. The pre-column deri - no adverse peripheral reactions were observed. The vatization procedure was carried out by mixing 100  µL HPLC method was modified to detect and measure six of sample and 100  µL of OPA reagent. The content was amino acid neurotransmitters in the same chromato- expressed as micromole of amino acid per gram of wet gram. Figure  1a indicates that 1  month after WBI, there tissue (mean ± SEM). was a significant decrease in GABA (p = 0.015), the main inhibitory neurotransmitter in the hypothalamus. The Brain receptors analysis levels of other inhibitory neurotransmitters such as Gly The expression of GABAa and GABAb receptors, and and Tau also decreased significantly (p = 0.04) in the NR1 and NR2A subunits of NMDA receptors was quan- hypothalamus compared to those detected in sham rats tified by Western-Blott according to a method previously (Fig.  1a). Similarly, we observed a significant decrease of reported [25]. Briefly, the frozen samples from the hypo - the excitatory amino acid Asp (p = 0.05) (Fig.  1a). In the thalamus, prefrontal cortex, and brainstem were homog- hypothalamus, we also observed that the amino acids enized in RIPA buffer containing a cocktail of protease with the highest concentration were Glu > Gln > GABA. inhibitors (Sigma Aldrich, USA). Homogenates were cen- On the contrary, the Gly content was the lowest (Fig. 1a). trifuged at 10,000×g for 10  min at 4  °C, and the super- We further examined the Gln/Glu and Glu/GABA ratios natants recovered. Protein concentration was measured since the balance between inhibition and excitation is using the BCA method. An aliquot was mixed with Lae- essential for the neurotransmission in the brain. There - mmli sample buffer and denatured at 100  °C for 5  min. fore, radiation did not induce significant changes in the Polyacrylamide gels (10%) were loaded with 50 µg of pro- Gln/Glu ratio (Fig.  1b); however, there was a significant tein. Proteins were transferred onto nitrocellulose mem- increase of the Glu/GABA ratio in the hypothalamus branes (Bio-Rad, USA) and then blocked with 5% nonfat after WBI (p = 0.004) (Fig. 1c). dry milk diluted in TBST for one h at room temperature. By contrast, in the prefrontal cortex, an alteration Next, membranes were incubated overnight at 4 °C with of the glutamatergic transmission was observed, since either anti-GABAb mouse monoclonal antibody (1:1000, excitatory neurotransmitters were increased 1  month SC-166408, Santa Cruz, USA), anti-GABAa mouse after treatment. Figure  2a illustrates how levels of Glu Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 4 of 13 Fig. 1 Neurotransmitters analysis in the hypothalamus 1 month after sham (white bars) or treatment with 11 Gy whole‑brain irradiation (black bars). The analyzed region is indicated in a Paxinos and Watson diagram [21]. a The concentration (μMol/g of fresh tissue) of the following amino acids: Asp, Glu, Gln, Gly, Tau, and GABA. Glutamine/Glutamate and Glutamate/GABA ratios are showed in b, c, respectively. Data are expressed as means ± SEM. Groups were statistically compared using the Mann–Whitney U test *p ≤ 0.05 F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 5 of 13 Fig. 2 Neurotransmitter levels in the prefrontal cortex 1 month after sham (white bars) or treatment with 11 Gy whole‑brain irradiation (black bars). The analyzed region is indicated in a Paxinos and Watson diagram [21]. a The concentration (μMol/g of fresh tissue) of the following amino acids: Asp, Glu, Gln, Gly, Tau, and GABA. Glutamine/Glutamate and Glutamate/GABA ratios are showed in b, c, respectively. Data are expressed as means ± SEM. Groups were statistically compared using the Mann–Whitney U test *p ≤ 0.05 Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 6 of 13 26–28]. Therefore, this dose radiation scheme is use - and Gln were significantly increased (p = 0.04, p = 0.004, ful in analyzing the substantial impairments associated respectively). with whole-brain irradiation. Compared with the other structures analyzed, we Our results indicate that WBI differentially modifies found that the prefrontal cortex contains the highest lev- the levels of some neurotransmitters and receptors in els of Glu and, on the contrary, the lowest levels of GABA the analyzed brain regions. GABA, the primary inhibi- (Fig. 2a). Figure 2b shows that WBI induced a significant tory neurotransmitter in the brain, was decreased in increase in the Gln/Glu ratio (p = 0.04), suggesting that the hypothalamus 1 month after WBI. It is well known the metabolism of Glu could be disrupted in the prefron- that inhibitory neurotransmission occurs through tal cortex of irradiated rats. Conversely, no differences GABA interaction with two classes of receptors, iono- were observed in the Glu/GABA ratio (Fig. 2c). tropic GABAa receptors and metabotropic GABAb In the brainstem, we detected that the amino acids receptors [29]. Remarkably, in this study, we showed with the highest concentration were Glu > Gln > Gly. In that WBI also prompted a reduction in the expression contrast, Tau was the least abundant. After performing of GABAa receptors in the hypothalamus. Studies have the statistical analysis, we noticed that both the amino proposed that the regulation of the stress response car- acid levels and the ratios remained without significant ried out by the HPA axis involves the participation of changes (Fig. 3a–c). GABAa receptors located in the hypothalamus [30]. One month after treatment, WBI also induced changes Consistently, infant female rats irradiated with a lower in the protein expression of some inhibitory and excita- dose (5  Gy) showed a reduction in the hypothalamic tory receptors. In the hypothalamus, the expression of levels of GABA [30]. The same infant rats also exhibited GABAa receptors was decreased significantly (p = 0.02) increased levels of the gonadotropic releasing hormone (Fig.  4a). A similar tendency was observed with GABAb; (GnRH) and precocious puberty symptoms [31], sup- however, this effect was not significant (Fig.  4b). We ana- porting GABA’s relevance in the endocrine regulation lyzed different subunits of the NMDA receptor; never - of the HPA axis after cranial irradiation. theless, no NR-1 changes were distinguished in irradiated Likewise, low GABA levels in the hypothalamus are rats (Fig. 4c). Detection of the NR2A subunit in the hypo- associated with fatigue, a symptom commonly reported thalamus was shallow to raise comparisons, and we avoid after whole-brain radiotherapy [11]. Also, it has been further increasing the protein concentration to elude suggested that the negative modulation of the GABAa tubulin signal saturation. function stimulates the occurrence of chronic fatigue Mostly, the radiation upregulated the expression of syndrome [32]. Otherwise, fatigue has also been cor- some receptors in the prefrontal cortex. Specifically, related with increased levels of inflammatory brain the WBI with a single dose of 11  Gy induced a signifi - cytokines. Interestingly, we have previously reported cant increase of the GABAa receptor (p = 0.05) (Fig.  5a) high hypothalamic levels of inflammatory cytokine and NMDA receptor NR1 subunit (p = 0.05) (Fig.  5c). IL-1β after irradiation [20]. Therefore, correlations Conversely, no significant changes were detected when between inflammatory response and neurochemical we analyzed the expression of GABAb (Fig.  5b) and the changes occurring in the hypothalamus after brain irra- NMDA receptor NR2A subunit (Fig. 5d). diation should be further explored. Although we observed a decrease in the GABAa recep- Moreover, hypothalamic GABA also participates in tor in the brainstem, this was not significant (Fig.  6a). the regulation of feeding behavior [33]. For example, Likewise, the GABAb receptor showed no changes the administration of GABAa agonist muscimol into (Fig.  6b). We were unable to detect any evidence of hypothalamic nuclei stimulates feeding. This response expression of the NMDA subunits in the brainstem. was inhibited by GABAa antagonist bicuculline [34]. Therefore, declining the GABAergic neurotransmis - Discussion sion in the hypothalamus could be contributing to the Here we used an irradiation rodent model using a sin- decreased appetite and weight loss observed after WBI gle fraction (11  Gy) to resemble a biologically equiva- [11, 35–37]. Hypothalamic GABA neurons are also lent dose of 72  Gy that corresponds to a traditional known to regulate several physiological and behavioral scheme of whole-brain radiotherapy with 10 Fractions responses associated with anxiety and stress. Shekhar of 3 Gy using an α/β = 2 for the healthy tissue, accord- [38] showed that GABAergic activity inhibition in the ing to the linear-quadratic model. Similar dose schemes dorsomedial hypothalamic area elicited evident signs in rodents have proof to efficiently replicate the behav - of anxiety in rats. Therefore, we suggest that reduced ioral effects observed in patients after whole brain GABA activity in the hypothalamus could be associated radiotherapy, like somnolence and cognitive effects [20, to anxiety behaviors observed after WBI [39]. F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 7 of 13 Fig. 3 Neurotransmitters analysis in the brainstem 1 month after sham (white bars) or treatment with 11 Gy whole‑brain irradiation (black bars). The analyzed region is indicated in a Paxinos and Watson diagram [21]. a The concentration (μMol/g of fresh tissue) of the following amino acids: Asp, Glu, Gln, Gly, Tau, and GABA. Glutamine/Glutamate and Glutamate/GABA ratios are showed in b, c, respectively. Data are expressed as means ± SEM. Groups were statistically compared using the Mann–Whitney U test *p ≤ 0.05. No statistical differences were found between groups Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 8 of 13 Fig. 4 Expression of GABAa (a) GABAb (b), and NR1 subunit of the NMDA receptor (c). The protein expression was analyzed in the rat hypothalamus by Western Blott 1 month after sham (white bars) or whole‑brain irradiation with a single dose of 11 Gy (black bars). The expression of the NR2A subunit was below standardized detection (data not shown). Data are expressed as means ± SEM. *p < 0.05 compared with the sham group using the Mann–Whitney U test GABA release in the posterior hypothalamus and euthanized during the day, and incremented sleepiness GABAa activation have somnogenic effects [40]. Since has been previously observed during the dark phase. previous results have shown that WBI may induce Still, further experiments could give more informa- sleep, reduced expression in GABAa may seem con- tion about circadian variations in neurotransmitters tradictory [10, 20]. Nevertheless, we must remember in response to radiation and its correlation with sleep that for evaluating neurotransmitters, animals were disturbances. F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 9 of 13 Fig. 5 Expression of GABAa (a), GABAb (b), NR1 (c), and NR2A subunit of the NMDA receptor (d). The protein expression was analyzed in the rat prefrontal cortex by Western Blott 1 month after sham (white bars) or whole‑brain irradiation with a single dose of 11 Gy (black bars). Data are expressed as means ± SEM. *p < 0.05 compared with the sham group using the Mann–Whitney U test Interestingly, the observed reduction in GABA in the Glycine is an inhibitory neurotransmitter acting mainly hypothalamus led to a significant increase in the Glu/ in the brainstem and spinal cord. Nevertheless, in this GABA ratio, which points out the prevalence of excita- work, no changes in glycine levels were observed in the tory processes. Increased Glu/GABA ratio may lead brainstem. Glycine also acts as an excitatory modula- to neurotoxic effects as Glu/GABA ratio is commonly tor of the NMDA receptors [42]. The NMDA receptors increased in other brain damage models like traumatic are tetrameric complexes composed of obligatory NR1 brain injury and kindling epilepsy [15, 41]. subunits co assembled with different NR2 (A-D) and, Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 10 of 13 Fig. 6 Expression of GABAa (a) and GABAb (b). The protein expression was analyzed in the brainstem by Western Blott 1 month after sham (white bars) or whole‑brain irradiation with a single dose of 11 Gy (black bars). It was unable to detect any evidence of the NMDA subunits in the brainstem. Data are expressed as means ± SEM. *p < 0.05 compared with the sham group using the Mann–Whitney U test less commonly, NR3 (A-B). Glutamate binds to the NR2 seen after treatment [49, 50]. Low prolactin levels could site while glycine binds to the NR1 site of NMDA recep- also affect stress response since prolactin has shown to tors. After WBI, we found reduced levels of glycine in the reduce anxiety behavior, modulate neurogenesis, and hypothalamus. Previous studies reported that oral doses exert neuroprotection [51]. Additionally, taurine may act of glycine could improve sleep quality and reduce fatigue as an anti-inflammatory and promote the cognitive func - during the day, apparently through activation of NMDA tion [52, 53]. Accordingly, lower taurine levels found in receptors in the hypothalamic suprachiasmatic nucleus the hypothalamus could be contributing to endocrine [43, 44]. Additionally, glycine agonists have anxiolytic and cognitive secondary effects reported after radiation and pro-cognitive effects and reduce brain injury induced treatments. by IL-1β [45, 46]. After an increase of IL-1β in the rat Aspartate levels were also reduced in the hypothala- hypothalamus induced by the WBI [20], the decrease of mus after radiation. Aspartate is an excitatory amino acid glycine could be a damage mechanism induced by radia- highly abundant in the hypothalamus [54]. Unlike glu- tion that negatively influences the cellular homeostasis in tamate, aspartate is a selective agonist of NMDA at the the hypothalamus. NR2 binding site. In the hypothalamus, aspartate and The reduction of taurine levels found in this study is NMDA have been implicated in the regulation of hor- consistent with previous endocrine hypothalamic effects monal release. Treatment with NMDA leads to enhanced also reported after WBI. Taurine is mainly produced by prolactin and growth hormone secretion [54]. There - astrocytes and with a high concentration found in the fore, similarly to taurine, low levels of aspartate could hypothalamus [47]. Taurine microinjections in the hypo- be implicated in endocrine disturbances observed after thalamic arcuate nucleus can stimulate prolactin pro- WBI. duction in the pituitary gland [48]. Consistently with WBI also modifies the prefrontal cortex inducing the reduced taurine levels, low prolactin concentrations higher levels of the excitatory neurotransmitters glu- have been reported after cranial irradiation in a rat model tamate and glutamine and a higher Gln/Glu ratio. The [37]. Furthermore, some children subjected to WBI have prefrontal cortex is known to be essential for higher cog- hypoprolactinemia, which have been correlated with nitive functions. After brain irradiation, higher glutamate lower growth hormone (GH) levels, a frequent sequel levels have been previously reported in other structures F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 11 of 13 like the striatum [55]. Glutamate regulates synaptic trans- focused radiation may depend on the specific irradiated mission and plasticity by activating ionotropic (AMPA brain region and the received dose. For instance, in Ste- and NMDA) and metabotropic receptors (mGluR1-R8). reotactic Radiotherapy, observed late behavioral effects Glutamate receptors overstimulation is known to induce are closely related to the dose spatial distribution and the potential damage in neural cells due to calcium overload dose received by specific regions [63]. Depending on dose [56]. Glutamine is transported from astrocytes into neu- distribution, even certain focused schemes can result in rons where glutaminase deaminates glutamine to pro- no behavioral changes [64]. Additionally, extreme incre- duce glutamate [57]. ments to conventional dose rates, like experimental Consequently, an increased glutamine/glutamate ratio ultra-high dose rate schemes (flash), have been shown has been proposed to be associated with decreased glial to reduce undesirable effects after whole-brain irradia - function or dysfunction of glia–neuron communica- tion [27]. Therefore, future work can focus on evaluat - tion [16]. Models of traumatic brain injury have also ing regional neurochemical changes for different dose shown increases in glutamate and glutamine and imbal- schemes. ances in glutamate-glutamine/GABA [15]. In patients with schizophrenia, it has been reported that glutamine and glutamine/glutamate ratio is increased in the medial Conclusion prefrontal cortex, which has been correlated with cog- Results illustrate how WBI modifies differentially amino nitive dysfunction [16]. Besides, glutamate increases in acid levels according to the analyzed brain region. At the prefrontal cortex of healthy patients have been cor- clinically equivalent doses, WBI can distinctively change related with cognitive and social dysfunctions [58]. In neurotransmitters and receptors in the brainstem, hypo- patients with diabetes type I, prefrontal glutamate-glu- thalamus, and the prefrontal cortex. At the hypothala- tamine–gamma-aminobutyric acid (Glx) was increased mus, WBI decreases the concentration of inhibitory and correlated with lower cognitive performance and neurotransmitters and receptors while at the prefrontal mild depression [59]. These studies demonstrate that dif - cortex increase excitatory neurotransmitters and recep- ferent pathophysiological conditions cause an imbalance tors. On the contrary, no changes were observed in of excitatory neurotransmission in the prefrontal cortex the brainstem. Increments in Glutamate/GABA in the and the concurrent appearance of cognitive impairments. hypothalamus and Glutamine/Glutamate in the frontal Interestingly, these two abnormalities can occur after cortex indicate modified neurochemical balance after WBI. Moreover, in this work, we also reported increased irradiation. GABAa receptors in the prefrontal cortex after brain We propose that observed changes could have an irradiation. Similar increases in prefrontal cortex GABAa essential role in the etiology of the side effects after receptors have been observed with aging [60]. Further, in WBI and suggest new prospects for therapeutic targets. hippocampal slices, irradiation increases the expression Hence, further studies should consider evaluations in of GABAa, correlating with long-term potentiation (LTP) both the hypothalamus and prefrontal cortex to better inhibition, which could be a mechanism involved in cog- understand the involved mechanisms in radiotherapy- nitive deficit [6]. induced brain injury. Lastly, we reported that WBI increased the expression of the NR1 subunit of the NMDA receptor in the pre- Abbreviations frontal cortex. Liang et  al., using a higher dose of radia- BED: Biologically effective dose; Fx: Fractions; RTOG: Radiation Therapy Oncol‑ tion (30  Gy), described the increased expression of NR1 ogy Group; HI: Homogeneity index defined as I /RI, where, I = maximum max max isodose in the target, and RI = reference isodose; GABA: Gamma‑aminobutyric and NR2A in the cortex, 1 and 2 months after irradiation acid; WBI: Whole‑brain irradiation; NMDA: N‑methyl‑D ‑aspartate; Gly: Glycine; [14]. Observed dissimilarities in NR2A could be associ- Tau: Taurine; Asp: Aspartate; Glu: Glutamate; Gln: Glutamine; RSS: Radiation ated with differences in dose escalation or coverage. The somnolence syndrome; HPA: Hypothalamic–pituitary–adrenal; HPLC: High‑ performance liquid chromatography; OPA: Ortho‑phthalaldehyde; RIPA: Radio ‑ functional significance of the change in NR1 expression immunoprecipitation assay; BCA: Bicinchonic acid; TBST: Tris buffered saline could be related to behavioral disturbances. The dele - plus 0.1% tween 20; IgG: Immunoglobulin G; HRP: Horseradish peroxidase; tion of NR1 has been shown to stimulate social behavior SPSS: Statistical package for the social sciences; GnRH: Gonadotropic releasing hormone; IL‑1β: Interleukin 1 beta; MRS: Magnetic resonance spectroscopy; [61]; by contrast, Iwata et al. demonstrated that radiation AMPA: α‑Amino ‑3‑hydroxy‑5‑methyl‑4‑isoxazolepropionic acid; Glx: Gluta‑ decreases the social interaction [62]. Thus, it seems plau - mate + glutamine + gamma‑aminobutyric acid; LTP: Long‑term potentiation. sible that WBI inhibits social behavior by increasing NR1 Acknowledgements expression. We want to thank reviewers for their valuable coments, to Dr. Sergio Moreno, Care should be taken when extrapolating these results chair of the radiosurgery department, and Dr. Jose Manuel Lárraga, chair of on radiation schemes other than WBI, as for brain Ste- Medical Physics, for their support. We also want to thank to Cynthia Lima Cruz for her valuable comments. reotactic Radiotherapy. The neurochemical effects of Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 12 of 13 Authors’ contributions plays a role in radiation‑induced brain injury. Radiat Res. 2014;181(1):21– JFP and JSH performed western‑blotting, analyzed, and interpreted the results. 32. https ://doi.org/10.1667/RR134 75.1. SM performed HPLC, analyzed, and interpreted the results. PB designed this 10. Ballesteros‑Zebadúa P, Chavarria A, Celis MA, Paz C, Franco ‑Pérez J. study, performed all irradiations, and was a major contributor in writing the Radiation‑induced neuroinflammation and radiation somnolence syn‑ manuscript. All authors read and approved the final manuscript. drome. CNS Neurol Disord Drug Targets. 2012;11(7):937–49. https ://doi. org/10.2174/18715 27311 20107 0937. Funding 11. Wong E, Rowbottom L, Tsao M, et al. Correlating symptoms and their This work was supported by the Mexican Council of Science CONACyT‑Mexico changes with survival in patients with brain metastases. Ann Palliat Med. (Fondo sectorial de investigación para la educación) Grant No. CB‑258296. 2016;5(4):253–66. https ://doi.org/10.21037 /apm.2016.09.01. 12. Mehta P, Fahlbusch FB, Rades D, Schmid SM, Gebauer J, Janssen S. Are Availability of data and materials hypothalamic‑pituitary (HP) axis deficiencies after whole brain radiother ‑ The datasets supporting the conclusions of this article are included within the apy ( WBRT ) of relevance for adult cancer patients? A systematic review article and its additional files. They are also available from the corresponding of the literature. BMC Cancer. 2019;19(1):1213. https ://doi.org/10.1186/ author on reasonable request.s1288 5‑019‑6431‑5. 13. Fan XW, Wang JQ, Wu JL, Wang HB, Wu KL. Simultaneously avoiding Ethics approval and consent to participate the hippocampus and hypothalamic‑pituitary axis during whole brain The CICUAL (Internal Committee for the Care and Use of Laboratory Animals) radiotherapy: a planning study. Med Dosim. 2019;44(2):130–5. https ://doi. of the National Institute of Neurology and Neurosurgery (INNN) have reviewed org/10.1016/j.meddo s.2018.04.004. and approved all the procedures included in this work. 14. Liang CD, Li WL, Liu N, Yin Y, Hao J, Zhao WQ. Eec ff ts of gamma knife irradiation on the expression of NMDA receptor subunits in rat forebrain. Consent for publication Neurosci Lett. 2008;439(3):250–5. https ://doi.org/10.1016/j.neule Not applicable. t.2008.05.046. 15. Amorini AM, Lazzarino G, Di Pietro V, et al. Severity of experimental trau‑ Competing interests matic brain injury modulates changes in concentrations of cerebral free The authors declare that they have no competing interests. amino acids. 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Whole-brain irradiation differentially modifies neurotransmitters levels and receptors in the hypothalamus and the prefrontal cortex

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Abstract

Background: Whole‑brain radiotherapy is a primary treatment for brain tumors and brain metastasis, but it also induces long‑term undesired effects. Since cognitive impairment can occur, research on the etiology of secondary effects has focused on the hippocampus. Often overlooked, the hypothalamus controls critical homeostatic functions, some of which are also susceptible after whole‑brain radiotherapy. Therefore, using whole ‑brain irradiation ( WBI) in a rat model, we measured neurotransmitters and receptors in the hypothalamus. The prefrontal cortex and brainstem were also analyzed since they are highly connected to the hypothalamus and its regulatory processes. Methods: Male Wistar rats were exposed to WBI with 11 Gy (Biologically Eec ff tive Dose = 72 Gy). After 1 month, we evaluated changes in gamma‑aminobutyric acid (GABA), glycine, taurine, aspartate, glutamate, and glutamine in the hypothalamus, prefrontal cortex, and brainstem according to an HPLC method. Ratios of Glutamate/GABA and Glutamine/Glutamate were calculated. Through Western Blott analysis, we measured the expression of GABAa and GABAb receptors, and NR1 and NR2A subunits of NMDA receptors. Changes were analyzed comparing results with sham controls using the non‑parametric Mann–Whitney U test (p < 0.05). Results: WBI with 11 Gy induced significantly lower levels of GABA, glycine, taurine, aspartate, and GABAa receptor in the hypothalamus. Also, in the hypothalamus, a higher Glutamate/GABA ratio was found after irradiation. In the prefrontal cortex, WBI induced significant increases of glutamine and glutamate, Glutamine/Glutamate ratio, and increased expression of both GABAa receptor and NMDA receptor NR1 subunit. The brainstem showed no statistically significant changes after irradiation. Conclusion: Our findings confirm that WBI can affect rat brain regions differently and opens new avenues for study. After 1 month, WBI decreases inhibitory neurotransmitters and receptors in the hypothalamus and, conversely, increases excitatory neurotransmitters and receptors in the prefrontal cortex. Increments in Glutamate/GABA in the hypothalamus and Glutamine/Glutamate in the frontal cortex indicate a neurochemical imbalance. Found changes could be related to several reported radiotherapy secondary effects, suggesting new prospects for therapeutic targets. Keywords: Whole brain irradiation, Amino acids, Neurotransmitters, Prefrontal cortex, Hypothalamus, GABAa, GABAb, NR1, NR2A Background *Correspondence: paolabaze@gmail.com Whole-brain radiotherapy is a primary medical treat- Laboratory of Medical Physics, National Institute of Neurology ment for some types of brain cancer, especially for brain and Neurosurgery, INNN, Insurgentes Sur 3877, Col. La Fama, C.P. 14269 Mexico City, Mexico metastasis. Although it improves patient’s survival, it is Full list of author information is available at the end of the article © The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 2 of 13 irrefutable that its use has been associated with several for cognitive processes, sleep regulation, and neuroim- adverse effects. According to the time of appearance, mune stress response [18, 19]. these effects have been classified as acute (hours to days), The data presented here demonstrate that WBI pro - early-delayed (1–6 months), and late-delayed (more than duces early delayed effects in the expression of recep - 6  months after) [1]. It is currently well established that tors and neurotransmitters levels at the hypothalamus whole-brain irradiation (WBI) at therapeutic doses leads and prefrontal cortex. Its possible implications for future to an increased risk for late-delayed cognitive impair- research are discussed. ments [2]. The hippocampus is the main structure asso - ciated with memory and cognition, and thus the most Methods studied after brain irradiation. Authors have proposed Animals that the ablation of hippocampal neurogenesis plays a Twenty-four male Wistar rats (270–300  g) were used. crucial role in cognitive impairment after radiation [3, Animals were housed in a room with controlled tem- 4]. It has also been suggested that the cellular mecha- perature (22 ± 2  °C), light–dark cycle (12:12), and nism underlying cognitive deficit involves alterations in ad  libitum access to water and food. All animals were receptors related to synaptic plasticity. Therefore, using handled according to Mexican Official Norms for the in vitro and in vivo models, it has been shown that irradi- production, care, and use of laboratory animals (NOM- ation changes the expression of excitatory and inhibitory 062-Z00-1999). Additionally, the Guide for the Care and receptors and neurotransmitters in the hippocampus Use of Laboratory Animals (NIH Guide) was revised and [5–9]. used as guidelines. WBI induces a complex initial cascade of neurochemi- cal processes, which can trigger the appearance of early- Whole‑brain irradiation delayed effects often considered transient and clinically The irradiation was performed using a micro-multileaf overlooked. Early-delayed effects include Radiation Som - collimator coupled to a linear accelerator for head treat- nolence Syndrome (RSS) characterized by increased sleep ments (Novalis Varian, 6 MV), as previously reported during the day, fatigue, decreased appetite, and weight [20]. A prescription dose of 11 Gy was chosen since it cor- loss [10]. Indeed, fatigue, loss of appetite, and weakness responds to a BED (Biologically effective dose) of 72  Gy are the most frequent symptoms reported to worsen in similar to the one used clinically (10 Fx, 3 Gy α/β = 2 Gy). patients after WBI [11]. Many of these symptoms are All animals were deeply anesthetized by administering closely related to alterations of the hypothalamic func- a mixture of ketamine (100  mg/kg) as a sedative agent, tion. Moreover, it has been observed that endocrine dis- and xylazine (10  mg/kg) as a muscle relaxant. Once the ruption of the hypothalamic–pituitary–adrenal (HPA) animals showed sensory stimulation response inhibition, axis can frequently appear after WBI [12]. The hypo - they were immobilized in a custom device and fixed to thalamus is a region that controls many critical homeo- the treatment table. A single dose of the drugs was suf- static functions, including those that are perturbed after ficient to carry out the WBI. The dynamic arcs tech - whole-brain radiotherapy [12, 13]. Therefore, it is rel - nique was employed at a dose rate of 500  UM/min. As evant to analyze hypothalamic neurochemical changes with patients, homogenous coverage was achieved all after WBI. Consequently, in this work, we measured over the brain (RTOG homogeneity index HI = 1.3) while excitatory and inhibitory neurotransmitters levels in the brain surrounding structures were protected. Sham ani- hypothalamus. Additionally, we measured the expression mals were mounted but received no dose. We used rat of gamma-aminobutyric acid (GABA) receptors (GABAa CT images and the software iPlan (BrainLab Germany) and GABAb) and NMDA receptor subunits NR1 and for the treatment planning, and the dose verification was NR2A since they have previously shown variations after performed using Monte Carlo techniques [20]. different irradiation doses in other brain structures [5, 6, 14]. Moreover, we calculated Glutamate/GABA and Glu- Brain amino acids analysis tamine/Glutamate ratios as they have been used as mark- Rats were killed by decapitation 1 month after the whole ers of neurochemical brain balance [15, 16]. brain irradiation or sham manipulation. All the ani- The brain response to radiation fluctuates according mals were sacrificed at the light phase between 9:00 and to the analyzed region. Thus, Todorovic et  al. [17] dem - 10:00  a.m. to avoid circadian variations. Brain regions onstrated that the antioxidant response after radiation is dissection was based on previously published protocols lower in the hippocampus than the cerebral cortex. For [21, 22]. Briefly, after decapitation, we extracted the brain this reason, we also analyze regions such as the prefron- by opening the skull through the midline. To obtain the tal cortex and brainstem. Both structures are highly con- prefrontal cortex, we first separate the olfactory bulb. nected to the hypothalamus and are considered critical Later, with the help of a rat brain slicer matrix, we made F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 3 of 13 a 1-mm coronal cut to discard mainly the motor cor- monoclonal antibody (1:1000, sc-376282, Santa Cruz, tex, then we obtained another 1-mm coronal cut. In this USA), anti-NR1 monoclonal mouse antibody (1:200, slice, we took the genus corpus callosum and the cap- BML-SA493-0015, Enzo, USA) or anti-NR2A monoclo- sula externa as a reference to delimitate and separate the nal mouse antibody (1:200, sc-31540, Santa Cruz, USA) striatum and thus get the prefrontal cortex sample. After diluted in TBST containing 5% nonfat dry milk. After that, we placed the brain ventral side up and located the washing with TBST, membranes were incubated for 1  h optical chiasm and the midbrain in the anterior and pos- at room temperature with anti-mouse IgG HRP conju- terior parts. With the help of a spatula, we punctured gated (1:10000, SAB3701105-2, Sigma Aldrich, USA). around such anatomical structures, and thus we obtained Protein bands were observed by chemiluminescence the hypothalamus. Finally, the brainstem was dissected using Luminata Forte (Millipore, USA) and an imaging after the elimination of the cerebellum and the inferior system Fusion Solo S (Vilber, France). After detection, and superior colliculus. The brainstem portion included we submerged membranes in stripping buffer washed the medulla oblongata and pons. The brain regions were with TBST, blocked with TBST containing 5% nonfat quickly dissected using the illustrations and coordinates dry milk, and incubated with anti-α Tubulin monoclonal shown in Paxinos and Watson Atlas [23]. The samples mouse antibody (1:1000, sc23948, Santa Cruz, USA) as were stored at − 70  °C until later analysis. The aspartate protein loading reference. (Asp), GABA, glutamate (Glu), glutamine (Gln), gly- cine (Gly), and taurine (Tau) contents were determined Statistical analysis according to a method previously reported [24]. Briefly, We compared sham manipulation data with irradiated tissue was homogenized and centrifuged at 4000×g animals running the two independent samples non-para- for 10  min at 4  °C, and the supernatants were kept at metric Mann–Whitney U test in SPSS (v 20 IBM). Differ - − 70  °C until assayed. The amino acid content was ana - ences were considered significant if p ≤ 0.05. lyzed using a high-performance liquid chromatography (HPLC) system Agilent 1100 series (Agilent Technolo- gies) equipped with a fluorescence detector and Adsor - Results bosphere ortho-phthalaldehyde (OPA) column (Alltech). In the present study, Wistar rats were treated with a The mobile phase consisted of a 50-mM sodium acetate WBI protocol calculated to guarantee adjacent tissue buffer (pH 5.9) solution containing 1.5% tetrahydro - protection, especially the mucosal tract. Consequently, furan and HPLC-grade methanol. The pre-column deri - no adverse peripheral reactions were observed. The vatization procedure was carried out by mixing 100  µL HPLC method was modified to detect and measure six of sample and 100  µL of OPA reagent. The content was amino acid neurotransmitters in the same chromato- expressed as micromole of amino acid per gram of wet gram. Figure  1a indicates that 1  month after WBI, there tissue (mean ± SEM). was a significant decrease in GABA (p = 0.015), the main inhibitory neurotransmitter in the hypothalamus. The Brain receptors analysis levels of other inhibitory neurotransmitters such as Gly The expression of GABAa and GABAb receptors, and and Tau also decreased significantly (p = 0.04) in the NR1 and NR2A subunits of NMDA receptors was quan- hypothalamus compared to those detected in sham rats tified by Western-Blott according to a method previously (Fig.  1a). Similarly, we observed a significant decrease of reported [25]. Briefly, the frozen samples from the hypo - the excitatory amino acid Asp (p = 0.05) (Fig.  1a). In the thalamus, prefrontal cortex, and brainstem were homog- hypothalamus, we also observed that the amino acids enized in RIPA buffer containing a cocktail of protease with the highest concentration were Glu > Gln > GABA. inhibitors (Sigma Aldrich, USA). Homogenates were cen- On the contrary, the Gly content was the lowest (Fig. 1a). trifuged at 10,000×g for 10  min at 4  °C, and the super- We further examined the Gln/Glu and Glu/GABA ratios natants recovered. Protein concentration was measured since the balance between inhibition and excitation is using the BCA method. An aliquot was mixed with Lae- essential for the neurotransmission in the brain. There - mmli sample buffer and denatured at 100  °C for 5  min. fore, radiation did not induce significant changes in the Polyacrylamide gels (10%) were loaded with 50 µg of pro- Gln/Glu ratio (Fig.  1b); however, there was a significant tein. Proteins were transferred onto nitrocellulose mem- increase of the Glu/GABA ratio in the hypothalamus branes (Bio-Rad, USA) and then blocked with 5% nonfat after WBI (p = 0.004) (Fig. 1c). dry milk diluted in TBST for one h at room temperature. By contrast, in the prefrontal cortex, an alteration Next, membranes were incubated overnight at 4 °C with of the glutamatergic transmission was observed, since either anti-GABAb mouse monoclonal antibody (1:1000, excitatory neurotransmitters were increased 1  month SC-166408, Santa Cruz, USA), anti-GABAa mouse after treatment. Figure  2a illustrates how levels of Glu Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 4 of 13 Fig. 1 Neurotransmitters analysis in the hypothalamus 1 month after sham (white bars) or treatment with 11 Gy whole‑brain irradiation (black bars). The analyzed region is indicated in a Paxinos and Watson diagram [21]. a The concentration (μMol/g of fresh tissue) of the following amino acids: Asp, Glu, Gln, Gly, Tau, and GABA. Glutamine/Glutamate and Glutamate/GABA ratios are showed in b, c, respectively. Data are expressed as means ± SEM. Groups were statistically compared using the Mann–Whitney U test *p ≤ 0.05 F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 5 of 13 Fig. 2 Neurotransmitter levels in the prefrontal cortex 1 month after sham (white bars) or treatment with 11 Gy whole‑brain irradiation (black bars). The analyzed region is indicated in a Paxinos and Watson diagram [21]. a The concentration (μMol/g of fresh tissue) of the following amino acids: Asp, Glu, Gln, Gly, Tau, and GABA. Glutamine/Glutamate and Glutamate/GABA ratios are showed in b, c, respectively. Data are expressed as means ± SEM. Groups were statistically compared using the Mann–Whitney U test *p ≤ 0.05 Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 6 of 13 26–28]. Therefore, this dose radiation scheme is use - and Gln were significantly increased (p = 0.04, p = 0.004, ful in analyzing the substantial impairments associated respectively). with whole-brain irradiation. Compared with the other structures analyzed, we Our results indicate that WBI differentially modifies found that the prefrontal cortex contains the highest lev- the levels of some neurotransmitters and receptors in els of Glu and, on the contrary, the lowest levels of GABA the analyzed brain regions. GABA, the primary inhibi- (Fig. 2a). Figure 2b shows that WBI induced a significant tory neurotransmitter in the brain, was decreased in increase in the Gln/Glu ratio (p = 0.04), suggesting that the hypothalamus 1 month after WBI. It is well known the metabolism of Glu could be disrupted in the prefron- that inhibitory neurotransmission occurs through tal cortex of irradiated rats. Conversely, no differences GABA interaction with two classes of receptors, iono- were observed in the Glu/GABA ratio (Fig. 2c). tropic GABAa receptors and metabotropic GABAb In the brainstem, we detected that the amino acids receptors [29]. Remarkably, in this study, we showed with the highest concentration were Glu > Gln > Gly. In that WBI also prompted a reduction in the expression contrast, Tau was the least abundant. After performing of GABAa receptors in the hypothalamus. Studies have the statistical analysis, we noticed that both the amino proposed that the regulation of the stress response car- acid levels and the ratios remained without significant ried out by the HPA axis involves the participation of changes (Fig. 3a–c). GABAa receptors located in the hypothalamus [30]. One month after treatment, WBI also induced changes Consistently, infant female rats irradiated with a lower in the protein expression of some inhibitory and excita- dose (5  Gy) showed a reduction in the hypothalamic tory receptors. In the hypothalamus, the expression of levels of GABA [30]. The same infant rats also exhibited GABAa receptors was decreased significantly (p = 0.02) increased levels of the gonadotropic releasing hormone (Fig.  4a). A similar tendency was observed with GABAb; (GnRH) and precocious puberty symptoms [31], sup- however, this effect was not significant (Fig.  4b). We ana- porting GABA’s relevance in the endocrine regulation lyzed different subunits of the NMDA receptor; never - of the HPA axis after cranial irradiation. theless, no NR-1 changes were distinguished in irradiated Likewise, low GABA levels in the hypothalamus are rats (Fig. 4c). Detection of the NR2A subunit in the hypo- associated with fatigue, a symptom commonly reported thalamus was shallow to raise comparisons, and we avoid after whole-brain radiotherapy [11]. Also, it has been further increasing the protein concentration to elude suggested that the negative modulation of the GABAa tubulin signal saturation. function stimulates the occurrence of chronic fatigue Mostly, the radiation upregulated the expression of syndrome [32]. Otherwise, fatigue has also been cor- some receptors in the prefrontal cortex. Specifically, related with increased levels of inflammatory brain the WBI with a single dose of 11  Gy induced a signifi - cytokines. Interestingly, we have previously reported cant increase of the GABAa receptor (p = 0.05) (Fig.  5a) high hypothalamic levels of inflammatory cytokine and NMDA receptor NR1 subunit (p = 0.05) (Fig.  5c). IL-1β after irradiation [20]. Therefore, correlations Conversely, no significant changes were detected when between inflammatory response and neurochemical we analyzed the expression of GABAb (Fig.  5b) and the changes occurring in the hypothalamus after brain irra- NMDA receptor NR2A subunit (Fig. 5d). diation should be further explored. Although we observed a decrease in the GABAa recep- Moreover, hypothalamic GABA also participates in tor in the brainstem, this was not significant (Fig.  6a). the regulation of feeding behavior [33]. For example, Likewise, the GABAb receptor showed no changes the administration of GABAa agonist muscimol into (Fig.  6b). We were unable to detect any evidence of hypothalamic nuclei stimulates feeding. This response expression of the NMDA subunits in the brainstem. was inhibited by GABAa antagonist bicuculline [34]. Therefore, declining the GABAergic neurotransmis - Discussion sion in the hypothalamus could be contributing to the Here we used an irradiation rodent model using a sin- decreased appetite and weight loss observed after WBI gle fraction (11  Gy) to resemble a biologically equiva- [11, 35–37]. Hypothalamic GABA neurons are also lent dose of 72  Gy that corresponds to a traditional known to regulate several physiological and behavioral scheme of whole-brain radiotherapy with 10 Fractions responses associated with anxiety and stress. Shekhar of 3 Gy using an α/β = 2 for the healthy tissue, accord- [38] showed that GABAergic activity inhibition in the ing to the linear-quadratic model. Similar dose schemes dorsomedial hypothalamic area elicited evident signs in rodents have proof to efficiently replicate the behav - of anxiety in rats. Therefore, we suggest that reduced ioral effects observed in patients after whole brain GABA activity in the hypothalamus could be associated radiotherapy, like somnolence and cognitive effects [20, to anxiety behaviors observed after WBI [39]. F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 7 of 13 Fig. 3 Neurotransmitters analysis in the brainstem 1 month after sham (white bars) or treatment with 11 Gy whole‑brain irradiation (black bars). The analyzed region is indicated in a Paxinos and Watson diagram [21]. a The concentration (μMol/g of fresh tissue) of the following amino acids: Asp, Glu, Gln, Gly, Tau, and GABA. Glutamine/Glutamate and Glutamate/GABA ratios are showed in b, c, respectively. Data are expressed as means ± SEM. Groups were statistically compared using the Mann–Whitney U test *p ≤ 0.05. No statistical differences were found between groups Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 8 of 13 Fig. 4 Expression of GABAa (a) GABAb (b), and NR1 subunit of the NMDA receptor (c). The protein expression was analyzed in the rat hypothalamus by Western Blott 1 month after sham (white bars) or whole‑brain irradiation with a single dose of 11 Gy (black bars). The expression of the NR2A subunit was below standardized detection (data not shown). Data are expressed as means ± SEM. *p < 0.05 compared with the sham group using the Mann–Whitney U test GABA release in the posterior hypothalamus and euthanized during the day, and incremented sleepiness GABAa activation have somnogenic effects [40]. Since has been previously observed during the dark phase. previous results have shown that WBI may induce Still, further experiments could give more informa- sleep, reduced expression in GABAa may seem con- tion about circadian variations in neurotransmitters tradictory [10, 20]. Nevertheless, we must remember in response to radiation and its correlation with sleep that for evaluating neurotransmitters, animals were disturbances. F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 9 of 13 Fig. 5 Expression of GABAa (a), GABAb (b), NR1 (c), and NR2A subunit of the NMDA receptor (d). The protein expression was analyzed in the rat prefrontal cortex by Western Blott 1 month after sham (white bars) or whole‑brain irradiation with a single dose of 11 Gy (black bars). Data are expressed as means ± SEM. *p < 0.05 compared with the sham group using the Mann–Whitney U test Interestingly, the observed reduction in GABA in the Glycine is an inhibitory neurotransmitter acting mainly hypothalamus led to a significant increase in the Glu/ in the brainstem and spinal cord. Nevertheless, in this GABA ratio, which points out the prevalence of excita- work, no changes in glycine levels were observed in the tory processes. Increased Glu/GABA ratio may lead brainstem. Glycine also acts as an excitatory modula- to neurotoxic effects as Glu/GABA ratio is commonly tor of the NMDA receptors [42]. The NMDA receptors increased in other brain damage models like traumatic are tetrameric complexes composed of obligatory NR1 brain injury and kindling epilepsy [15, 41]. subunits co assembled with different NR2 (A-D) and, Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 10 of 13 Fig. 6 Expression of GABAa (a) and GABAb (b). The protein expression was analyzed in the brainstem by Western Blott 1 month after sham (white bars) or whole‑brain irradiation with a single dose of 11 Gy (black bars). It was unable to detect any evidence of the NMDA subunits in the brainstem. Data are expressed as means ± SEM. *p < 0.05 compared with the sham group using the Mann–Whitney U test less commonly, NR3 (A-B). Glutamate binds to the NR2 seen after treatment [49, 50]. Low prolactin levels could site while glycine binds to the NR1 site of NMDA recep- also affect stress response since prolactin has shown to tors. After WBI, we found reduced levels of glycine in the reduce anxiety behavior, modulate neurogenesis, and hypothalamus. Previous studies reported that oral doses exert neuroprotection [51]. Additionally, taurine may act of glycine could improve sleep quality and reduce fatigue as an anti-inflammatory and promote the cognitive func - during the day, apparently through activation of NMDA tion [52, 53]. Accordingly, lower taurine levels found in receptors in the hypothalamic suprachiasmatic nucleus the hypothalamus could be contributing to endocrine [43, 44]. Additionally, glycine agonists have anxiolytic and cognitive secondary effects reported after radiation and pro-cognitive effects and reduce brain injury induced treatments. by IL-1β [45, 46]. After an increase of IL-1β in the rat Aspartate levels were also reduced in the hypothala- hypothalamus induced by the WBI [20], the decrease of mus after radiation. Aspartate is an excitatory amino acid glycine could be a damage mechanism induced by radia- highly abundant in the hypothalamus [54]. Unlike glu- tion that negatively influences the cellular homeostasis in tamate, aspartate is a selective agonist of NMDA at the the hypothalamus. NR2 binding site. In the hypothalamus, aspartate and The reduction of taurine levels found in this study is NMDA have been implicated in the regulation of hor- consistent with previous endocrine hypothalamic effects monal release. Treatment with NMDA leads to enhanced also reported after WBI. Taurine is mainly produced by prolactin and growth hormone secretion [54]. There - astrocytes and with a high concentration found in the fore, similarly to taurine, low levels of aspartate could hypothalamus [47]. Taurine microinjections in the hypo- be implicated in endocrine disturbances observed after thalamic arcuate nucleus can stimulate prolactin pro- WBI. duction in the pituitary gland [48]. Consistently with WBI also modifies the prefrontal cortex inducing the reduced taurine levels, low prolactin concentrations higher levels of the excitatory neurotransmitters glu- have been reported after cranial irradiation in a rat model tamate and glutamine and a higher Gln/Glu ratio. The [37]. Furthermore, some children subjected to WBI have prefrontal cortex is known to be essential for higher cog- hypoprolactinemia, which have been correlated with nitive functions. After brain irradiation, higher glutamate lower growth hormone (GH) levels, a frequent sequel levels have been previously reported in other structures F ranco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 11 of 13 like the striatum [55]. Glutamate regulates synaptic trans- focused radiation may depend on the specific irradiated mission and plasticity by activating ionotropic (AMPA brain region and the received dose. For instance, in Ste- and NMDA) and metabotropic receptors (mGluR1-R8). reotactic Radiotherapy, observed late behavioral effects Glutamate receptors overstimulation is known to induce are closely related to the dose spatial distribution and the potential damage in neural cells due to calcium overload dose received by specific regions [63]. Depending on dose [56]. Glutamine is transported from astrocytes into neu- distribution, even certain focused schemes can result in rons where glutaminase deaminates glutamine to pro- no behavioral changes [64]. Additionally, extreme incre- duce glutamate [57]. ments to conventional dose rates, like experimental Consequently, an increased glutamine/glutamate ratio ultra-high dose rate schemes (flash), have been shown has been proposed to be associated with decreased glial to reduce undesirable effects after whole-brain irradia - function or dysfunction of glia–neuron communica- tion [27]. Therefore, future work can focus on evaluat - tion [16]. Models of traumatic brain injury have also ing regional neurochemical changes for different dose shown increases in glutamate and glutamine and imbal- schemes. ances in glutamate-glutamine/GABA [15]. In patients with schizophrenia, it has been reported that glutamine and glutamine/glutamate ratio is increased in the medial Conclusion prefrontal cortex, which has been correlated with cog- Results illustrate how WBI modifies differentially amino nitive dysfunction [16]. Besides, glutamate increases in acid levels according to the analyzed brain region. At the prefrontal cortex of healthy patients have been cor- clinically equivalent doses, WBI can distinctively change related with cognitive and social dysfunctions [58]. In neurotransmitters and receptors in the brainstem, hypo- patients with diabetes type I, prefrontal glutamate-glu- thalamus, and the prefrontal cortex. At the hypothala- tamine–gamma-aminobutyric acid (Glx) was increased mus, WBI decreases the concentration of inhibitory and correlated with lower cognitive performance and neurotransmitters and receptors while at the prefrontal mild depression [59]. These studies demonstrate that dif - cortex increase excitatory neurotransmitters and recep- ferent pathophysiological conditions cause an imbalance tors. On the contrary, no changes were observed in of excitatory neurotransmission in the prefrontal cortex the brainstem. Increments in Glutamate/GABA in the and the concurrent appearance of cognitive impairments. hypothalamus and Glutamine/Glutamate in the frontal Interestingly, these two abnormalities can occur after cortex indicate modified neurochemical balance after WBI. Moreover, in this work, we also reported increased irradiation. GABAa receptors in the prefrontal cortex after brain We propose that observed changes could have an irradiation. Similar increases in prefrontal cortex GABAa essential role in the etiology of the side effects after receptors have been observed with aging [60]. Further, in WBI and suggest new prospects for therapeutic targets. hippocampal slices, irradiation increases the expression Hence, further studies should consider evaluations in of GABAa, correlating with long-term potentiation (LTP) both the hypothalamus and prefrontal cortex to better inhibition, which could be a mechanism involved in cog- understand the involved mechanisms in radiotherapy- nitive deficit [6]. induced brain injury. Lastly, we reported that WBI increased the expression of the NR1 subunit of the NMDA receptor in the pre- Abbreviations frontal cortex. Liang et  al., using a higher dose of radia- BED: Biologically effective dose; Fx: Fractions; RTOG: Radiation Therapy Oncol‑ tion (30  Gy), described the increased expression of NR1 ogy Group; HI: Homogeneity index defined as I /RI, where, I = maximum max max isodose in the target, and RI = reference isodose; GABA: Gamma‑aminobutyric and NR2A in the cortex, 1 and 2 months after irradiation acid; WBI: Whole‑brain irradiation; NMDA: N‑methyl‑D ‑aspartate; Gly: Glycine; [14]. Observed dissimilarities in NR2A could be associ- Tau: Taurine; Asp: Aspartate; Glu: Glutamate; Gln: Glutamine; RSS: Radiation ated with differences in dose escalation or coverage. The somnolence syndrome; HPA: Hypothalamic–pituitary–adrenal; HPLC: High‑ performance liquid chromatography; OPA: Ortho‑phthalaldehyde; RIPA: Radio ‑ functional significance of the change in NR1 expression immunoprecipitation assay; BCA: Bicinchonic acid; TBST: Tris buffered saline could be related to behavioral disturbances. The dele - plus 0.1% tween 20; IgG: Immunoglobulin G; HRP: Horseradish peroxidase; tion of NR1 has been shown to stimulate social behavior SPSS: Statistical package for the social sciences; GnRH: Gonadotropic releasing hormone; IL‑1β: Interleukin 1 beta; MRS: Magnetic resonance spectroscopy; [61]; by contrast, Iwata et al. demonstrated that radiation AMPA: α‑Amino ‑3‑hydroxy‑5‑methyl‑4‑isoxazolepropionic acid; Glx: Gluta‑ decreases the social interaction [62]. Thus, it seems plau - mate + glutamine + gamma‑aminobutyric acid; LTP: Long‑term potentiation. sible that WBI inhibits social behavior by increasing NR1 Acknowledgements expression. We want to thank reviewers for their valuable coments, to Dr. Sergio Moreno, Care should be taken when extrapolating these results chair of the radiosurgery department, and Dr. Jose Manuel Lárraga, chair of on radiation schemes other than WBI, as for brain Ste- Medical Physics, for their support. We also want to thank to Cynthia Lima Cruz for her valuable comments. reotactic Radiotherapy. The neurochemical effects of Franco‑Pérez et al. Radiat Oncol (2020) 15:269 Page 12 of 13 Authors’ contributions plays a role in radiation‑induced brain injury. Radiat Res. 2014;181(1):21– JFP and JSH performed western‑blotting, analyzed, and interpreted the results. 32. https ://doi.org/10.1667/RR134 75.1. SM performed HPLC, analyzed, and interpreted the results. PB designed this 10. Ballesteros‑Zebadúa P, Chavarria A, Celis MA, Paz C, Franco ‑Pérez J. study, performed all irradiations, and was a major contributor in writing the Radiation‑induced neuroinflammation and radiation somnolence syn‑ manuscript. All authors read and approved the final manuscript. drome. CNS Neurol Disord Drug Targets. 2012;11(7):937–49. https ://doi. org/10.2174/18715 27311 20107 0937. Funding 11. Wong E, Rowbottom L, Tsao M, et al. Correlating symptoms and their This work was supported by the Mexican Council of Science CONACyT‑Mexico changes with survival in patients with brain metastases. Ann Palliat Med. (Fondo sectorial de investigación para la educación) Grant No. CB‑258296. 2016;5(4):253–66. https ://doi.org/10.21037 /apm.2016.09.01. 12. Mehta P, Fahlbusch FB, Rades D, Schmid SM, Gebauer J, Janssen S. Are Availability of data and materials hypothalamic‑pituitary (HP) axis deficiencies after whole brain radiother ‑ The datasets supporting the conclusions of this article are included within the apy ( WBRT ) of relevance for adult cancer patients? A systematic review article and its additional files. They are also available from the corresponding of the literature. BMC Cancer. 2019;19(1):1213. https ://doi.org/10.1186/ author on reasonable request.s1288 5‑019‑6431‑5. 13. Fan XW, Wang JQ, Wu JL, Wang HB, Wu KL. 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