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Deep hypothermia prevents striatal alterations produced by perinatal asphyxia: Implications for the prevention of dyskinesia and psychosis

GABAergic medium spiny neurons are the main neuronal population in the striatum. Calbindin is preferentially expressed in medium spiny neurons involved in the indirect pathway. The aim of the present work is to analyze the effect of perinatal asphyxia on different subpopulations of GABAergic neurons in the striatum and to assess the outcome of deep therapeutic hypothermia. The uterus of pregnant rats was removed by cesarean section and the fetuses were exposed to hypoxia by immersion in water (19 min) at 37°C (perinatal asphyxia). The hypothermic group was exposed to 10°C during 30 min after perinatal asphyxia. The rats were euthanized at the age of one month (adolescent/adult rats), their brains were dissected out and coronal sections were immunolabeled for calbindin, calretinin, NeuN, and reelin. Reelin+ cells showed no staining in the striatum besides subventricular zone. The perinatal asphyxia (PA) group showed a significant decrease in calbindin neurons and a paradoxical increase in neurons estimated by NeuN staining. Moreover, calretinin+ cells, a specific subpopulation of GABAergic neurons, showed an increase caused by PA. Deep hypothermia reversed most of these alterations probably by protecting calbindin neurons. Similarly, there was a reduction of the diameter of the anterior commissure produced by the asphyxia that was prevented by hypothermic treatment.

has high density of calbindin+ neurons and it also represents one of the major pools of D2 receptor expression in the brain (Hurd, Suzuki, & Sedvall, 2001). This is important, not only because D2 antagonism is the therapeutic target of antipsychotics, but because this compounds are also used in the treatment of chorea/dyskinesia (reviewed by Coppen & Roos, 2017). There are also reports of cross-vulnerabilities for both pathologies (Kindler et al., 2016;Koning et al., 2011;Whitty, Owoeye, & Waddington, 2009).
The dorsal striatum is thought to be involved mostly in motor behaviors, while ventral striatum (or nucleus accumbens, NAcc) is crucial for motivational, cognitive, and emotional processes (Groenewegen, 2003;Robbins & Everitt, 1996).
Medium spiny neurons (MSN) are the large majority of striatal neurons, 95% in rodents. MSN are GABAergic neurons that can be separated into two subpopulations: the neurons of the direct pathway that mainly express dopamine D1 receptor and project to the internal globus pallidus, and the MSN of the indirect pathway that mostly express the dopamine D2 receptor and project to the lateral globus pallidus.
Nowadays the only available therapeutic approach to prevent the damage caused by PA is hypothermia. These therapies involve mild hypothermia over long periods of time and have proven to be effective in mitigating the damage caused by PA in animal models and in humans. Some procedures involve exposition to a cooled blanket until rectal temperature reaches 33.5 C for 72 hr. Significant improvements on survival and severe disabling sequelae have been reported under these conditions (Meloni, Mastaglia, & Knuckey, 2008;Simbruner et al., 2010).
In previous works from our group, deep hypothermia prevented histological alterations in medial prefrontal cortex and anterior insular cortex. PA decreased the density of calbindin+ neurons in the anterior insular cortex and deep hypothermia reversed this alteration. There were also behavioral alterations involving social interaction such as a decrease in play soliciting that also was reversed by deep hypothermia (Vázquez-Borsetti, Peña, Rojo, Acuña, & Loidl, 2018).
In order to deepen the knowledge about the mechanisms that generates dyskinesia and vulnerability to schizophrenia and to assess the therapeutic effect of hypothermia, we performed a set of immunohistochemical studies. We compared the densities of calbindin+, calretinin+ and NeuN+ cells among rats exposed to PA and PA+ therapeutic hypothermia. Calbindin is a good marker of MSN and it is distributed in the matrix of the striatum where the neurons preferentially participate in the indirect pathway (Bennett & Bolam, 1993). Calretinin is a marker of a specific subpopulation of GABAergic interneurons in the striatum (Petryszyn, Parent, & Parent, 2018). Finally, the neuronal nuclear protein (NeuN) is a widely used neuronal marker (Azevedo et al., 2009) and we combined it with DAPI that stains all cell nuclei.

| METHODS
PA was induced in Sprague-Dawley albino rats using a noninvasive hypoxia-ischemia model, as described before (Loidl et al., 1994).
Housing and in vivo experiments were performed in the animal facility of the Facultad de Medicina de la Universidad de Buenos Aires. Personnel of the animal house controlled the welfare of the animals daily.
The sick animals were euthanized or treated according to each case.
The animals were bred and handled in accordance with the guidelines published in the Guide for the Care and Use of Laboratory Animals CICUAL, and the principles presented in the "Guidelines for the Use of

| Treatment
Naive pregnant rats were euthanized by decapitation and were immediately hysterectomized after their first vaginal delivery. Term fetuses still in the womb were subjected to transient asphyxia by immersion of both uterine horns in a water bath for 19 min (18 0 30 00 to 19 0 30 00 ) at 37 C (PA and hypothermic group). After asphyxia, or immediately after hysterectomy in case of cesarean control (CC), the uterine horns were opened, pups were removed and stimulated to breathe. The pups were then placed for recovery under a heat lamp for an hour and were given to a surrogate mother. In the case of the group with hypothermia post asphyxia (HYPPA), pups were exposed to a temperature of 10 C for 30 min. At the end of the hypothermic shock, rectal temperature was measured with a thermocouple obtaining a temperature of 15 ± 1 C. To avoid the influence of hormonal changes, only male rats were included in this study. The pups were weaned on postnatal day 25; litters were divided by sex and, males from the same mother were housed together. Sham rats were also included as controls, vaginal control (VC).

| Histology and immunohistochemistry
In order to obtain the tissues for the immunohistochemical studies, either 3 or 4 rats, 40 days old, were selected within each experimental group (VC, CC and HYPPA, n = 3 while PA, n = 4). They were intraperitoneally anesthetized with an ip overdose of ketamine (250 mg/k) and xylazine 25 (mg/k) and perfused transcardially with 50 ml of saline solution, followed by 300 ml of fixative (4% paraformaldehyde) at room temperature. Brains were removed and stored for a period of time no longer than 2 days in phosphate buffered saline (PBS; pH 7.4; 4 C). Tissue sections of 40 μm of thickness were cut with a vibratome and stored in a cryoprotective solution at −20 C.
The sections were collected from rostral to caudal in multiwells. The first 10 sections from the beginning of the dorsal striatum were discarded and three wells per rat were filled sequentially with 15 slices each, starting around anteroposterior (AP) coordinate = 2.16 and ending on around AP = 0.36 (Paxinos & Watson, 2004). The start of the stria terminalis was used as landmark to stop collecting tissue ( Figure 1). One section from each well (three sections per rat) was preincubated for 30 min in 0.1 M PBS; 0.3% Triton X-100 and 1% goat serum at room temperature. Incubation with the primary antibody (1:1,000; mouse monoclonal, anti-reelin (Abcam), anti-calbindin (Leica), or anti-NeuN (Millipore) was performed during 3-4 days at 4 C in multiwells (see Table 1 for more information about the antibodies). Subsequently, sections were incubated for 120 min with biotinylated secondary antibody anti-mouse (1:1000, Thermo Fisher Scientific) followed by 30 min in Vectastain Elite ABC solution (Vector Laboratories). Sections were immersed in a solution of 0.05 mg/ml 3,3 0 -diaminobenzidine of containing 2 μl/ml of H 2 O 2 and nickel ammonium until color development (in all cases less than 10 min). The reaction was stopped by performing two rinses in distilled H 2 O.
In the case of fluorescent stain, the sections were incubated with an anti-mouse secondary antibody conjugated with Alexa 488 (Thermofisher Scientific, 1:1,000). The stained sections were mounted on gelatin-coated slides for microscopic analysis.

| Image analysis and cell counting
In order to improve consistency of the data and decrease the impact of possible methodological errors, immunohistochemical studies were performed at least 3 times including different animals of all groups by each assay, with final N = 3-4. Because there was some variability among assays, we decided to normalize the results to the VC group counts that were considered as 100%. Systematic random sampling was used to obtain unbiased estimates of cell densities. Equidistant counting frames were generated and randomly assigned inside the region of interest using the grid tool from the program ImageJ (FIJI distribution).
The dimensions of the counting frames were determined with the aim of counting more than 150 cells per region of interest (ROI) in each slice. The sizes of the counting frames were 40 × 40 μm for calbindin and 30 × 30 μm for DAPI and NEUN. The distances between frames were 280 μm for calbindin and 300 μm for DAPI and NEUN in the case of the dorsal striatum, and 40 and 30 μm, respectively, in the case of the nucleus accumbens.
The exception to this rule was the calretinin staining, due to the low number of cells we counted all cells inside the ROI. Manual counting was performed by a skilled counter blind to treatment using also ImageJ. All the cells inside the frame and in contact with two consecutive borders of the square were counted (but not the cells in contact with the other two borders). Within each experimental subject, three sections at different antero-posterior levels were analyzed. For DAPI, NeuN and Calbindin several images with ×10 magnification were assembled in a panoramic picture covering the dorsal striatum sampling area (Figure 1, red square). In the case of the calretinin immunostaining, the same analysis was performed but F I G U R E 1 Panoramic NeuN staining of the whole striatum, the red square indicates the sampling zone of the dorsal striatum, and the yellow line indicates the sampling zone of the nucleus accumbens, the white arrow indicates the beginning of the temporal arm of the anterior commissure used as landmark for the posterior end of the structure (a). There was no gross/evident morphological differences in the striatum and the width of the structure was consistent among treated and control groups with no significant differences (unpaired t test p = .16) (b). Approximated proportion of the different populations analyzed in this work (c). Dorsal striatum bar = 1 mm; Nucleus accumbens bar = 200 μm [Color figure can be viewed at wileyonlinelibrary.com] the panoramic composition was carried out with pictures of ×20 magnification. Cell densities were calculated by dividing the number of cells counted by the sampled area. In the case of the nucleus accumbens, the anterior commissure was used as landmark to determine the region of interest. Also the start of the stria terminalis was used as landmark for the end of the structure. The delimitation of this ROI was determined in each case following Paxinos atlas as reference (Paxinos & Watson, 2004), an example can be seen in Figure 1 (yellow line). In all cases the focal point was settled outside of a guard zone of 2 μm from the surface. The thickness of the slice analyzed was 8.5 μm.
Results were reported using one-way ANOVA and the multiple comparison test of Holm-Sidak for post hoc comparison. In all cases, the errors reported were standard errors. No outliers were excluded.
The Shapiro-Wilk test indicated no significant deviation from normality and the Brown-Forsythe test indicated homogeneity of variances.
Antibodies used in this study  These changes do not seem to be accompanied by changes in the gross morphology of the striate. We measured the width of the structure and it did not show differences between control and PA groups (unpaired t test p = .16; Figure 1). See Tables 2 and 3  immunohistochemical studies. Reelin labeling did not show any positive staining in the striatum apart from the subventricular zone where there was a group of cells with very strong labeling ( Figure 3).
Calretinin neurons showed strong staining with low density of neurons scattered in the structure. The cell densities in the control group were 0.40 ± 0.06 cells/0.01 mm 2 in the dorsal striatum and 0.44 ± 0.11 cells/0.01 mm 2 in the ventral striatum. The relationship of calbindin/calretinin cells in the area was greater than 50 to 1 (Figure 3).
In the dorsal striatum there was a significant increase of neurons with calretinin produced by asphyxia (126.6% ± 25.12) and deep hypothermia prevented this alteration (F(2, 6) = 13.52, p = .006; HYPPA vs. PA p = .021CC vs. PA p = .0047). In the case of NAcc, a similar pattern was observed but with no significant differences (F(2, 6) = 0.69, p = .53; Figure 3). See Table 2 for more information about statistical testing.

| Morphometric analysis of the anterior commissure
In order to analyze the health of the white matter a morphometric analysis of the anterior commissure was performed. The comparison of the nuclei density between groups did not show a significant difference (F(2, 6) = 0.58, p = .582). We also measured the thickness of the structure in the minor axis, a notorious shrinkage of the anterior commissure was produced by PA (−30.36% ± 8.39), this was reversed by deep hypothermia (F(2, 6) = 11.05, p = .009 HYPPA vs. PA p = .009 CC vs. PA p = .01). The VC values for these parameters were 0.325 ± 0.01 mm in the case of the minor axis of the anterior commissure and 63.83 ± 1.58 for DAPI cell counts ( Figure 6). See Table 5 for more information about statistical testing.

| DISCUSSION
In the present work we found that perinatal asphyxia induced significant morphological and histological alterations in the rat striatum.
These alterations were prevented by exposure to deep hypothermia.

| Calbindin
The decrease in neurons with calbindin produced by asphyxia is noteworthy. This phenomenon was widely observed previously by our group and elsewhere. Decrease of calbindin+ neurons has been reported in the striatum (George et al., 2007;Guan et al., 1999;Mallard, Waldvogel, Williams, Faull, & Gluckman, 1995;Van De Berg et al., 2003) in the prefrontal cortex (Vázquez-Borsetti et al., 2016) and in the anterior insular cortex (Vázquez-Borsetti et al., 2018) among others. Calbindin expressing neurons of the striatum correspond to a subpopulation of MSN, although there is also a small subpopulation that appears to be interneurons that do not have spines (Bennett & Bolam, 1993;Celio, 1990; We also found a relatively high proportion of neurons with calbindin compared to total neurons, estimated by NeuN. These results are not surprising if we take into consideration that 95 % of the striatal neurons correspond to medium-sized spiny neurons (Kemp & Powell, 1971).
The specific vulnerability of calbindin+ GABAergic neurons could be related, among others, to the excitatory-to-Inhibitory GABA switch that occurs at birth. During this period, It has been reported that interneurons are both; the source and the target of the first synapses, and glutamatergic transmission is mostly silent (Hennou, Khalilov, Diabira, Ben-Ari, & Gozlan, 2002). This excitatory GABAergic transmission generates giant depolarizing potentials (Ben-Ari, 2014). This phenomenon was observed in rodents and primates but there is evidence supporting similar process in humans. For example, GABAergic agonists generate epileptiform spasms at birth in rodents, primates and humans (Khazipov et al., 2001). Also, the use of GABAergic agonists in human newborns is highly contraindicated because they generate strong adverse and paradoxical effects (Chess & D'Angio, 1998;Ng et al., 2002).

| Hypothermia
An important characteristic of the therapeutic strategy used in this work is the period of exposition to hypothermia that is needed to accomplish its therapeutic effect. There is a relationship between the time of exposition and the final temperature needed to generate the therapeutic effect.
This treatment avoided the decrease in calbindin neuronal density caused by PA. Currently, the only therapeutic strategy available to treat perinatal asphyxia involves mild hypothermia, approximately 33 C for 72 hr (Simbruner et al., 2010). We believe that it is possible to reduce the time of exposure to hypothermia by using lower temperatures. Moreover, we think that lower temperatures as the ones used in this work could produce a stronger therapeutic effect. In previous studies with the same conditions, we saw that the exposure to a  (George et al., 2007).
Input from deep hypothermia during aortic arch surgery indicates that the great majority of adults and children can be supported unharmed under a circulatory arrest by 30 min at 18 C (Fleck et al., 2003;Parissis, Hamid, Soo, & Al-Alao, 2011).
The mechanism involved in the protective effect of hypothermia is more complex than a simple reduction in metabolism. Hypothermia is especially good to protect from excitotoxic damage (Leaw et al., 2017). Probably the mechanisms include: (a) Reduction of neuronal firing. (b) Restoration of the resting membrane potential (Thompson, Masukawa, & Prince, 1985). (c) Reduction of metabolism during the reuptake of the intracellular calcium. (d) Reuptake of neurotransmitters from the synaptic cleft.
In a previous work we reported that hypothermia during asphyxia does not prevent the deterioration in social interaction produced by PA, but it was therapeutic when administered after asphyxia (Vázquez-Borsetti et al., 2018). Although, it is well known that hypothermia during asphyxia has a strong protective effect (Loidl et al., 1998), this injury is characterized by two phases of damage. One phase takes place during asphyxia and the other takes place during reperfusion. It appears that social interaction is more vulnerable to reperfusion injury. The three main factors linked to this damage are: excitotoxic damage, inflammation and oxidative damage by reactive oxygen species.
Hypothermia generates a decrease in available oxygen that can be beneficial in the case of oxidative damage. In this sense, it is important not to generate a forced rewarming, but rather to allow the body to recover slowly the temperature (Scaravilli, Bonacina, & Citerio, 2012;Wong, 1983). Finally, hypothermia is also very well known for having anti-inflammatory properties (Zhao et al., 2019).

| Increase in calretinin, NeuN and DAPI
Perinatal asphyxia increases the density of neurons with calretinin in the striatum. Although this result seems paradoxical, it has already been reported by other group (Yang, You, & Levison, 2008). We hypothesize that this increase is caused by a compensatory effect exerted by the loss of calbindin neurons. The fact that deep hypothermia also prevents this alteration supports this interpretation.
The neurons with calretinin in the striatum are interneurons (Petryszyn et al., 2018) then, a priori, they could not correct the damage in the indirect pathway.
PA produced a slight but significant increase in NeuN density. Cell density estimated by DAPI staining followed the same pattern without reaching significant statistical differences. Other important piece of information is that the morphometry did not show any contraction/ shrinkage of the structure. Human and rat brains continue to grow after birth (Anon, 1984;Fish & Winick, 1969;Ortinau et al., 2018). The loss of calbindin cells leaves space that could be occupied by other neurons.
If the cell body and neuropil of the new neurons use less space than the former neurons, an increase in cell density seems plausible.
Another factor that may be contributing to this result is a homeostatic increase in other cell types to maintain the inhibitory balance of the structure. In previous studies we have found similar results in the anterior insular cortex with reelin-calretinin neurons. The increase in calretinin observed in this work is not enough to explain the compensation/increase in NeuN. Then, we predict increased cell density of other neuronal subpopulations in the striatum. If some of these neurons belong to the direct pathway, the imbalance between the direct and indirect pathway would be even greater than expected. This could reduce even further the inhibitory capacity of the striatum and increase the effect of dopamine in the structure worsening the symptoms of dyskinesia and psychosis. This point of view, is supported by previous works where PA produced an increase in the number of cells labeled using antibodies against neuronal nitric oxide synthase and cyclic guanosine monophosphate in the striatum (Barkhuizen, Van De Berg, et al., 2017). This is consistent with previous related research in the same model of asphyxia, where an increased striatal cGMP production was found at P10 (Loidl et al., 1998). The nitric oxide synthase immunoreactive neurons where reported to express dopamine D1 receptor (and/or D5) and partially co-localize with parvalbumin neurons. The cGMP+ neurons match the MSN subpopulation (Arcangeli et al., 2013;Ariano, 1983).
Additionally, an interesting finding was the presence of reelin+ cells in the subventricular zone but not in the rest of striatum.
As far as we know, this is the first time that the presence of reelin in the subventricular zone has been reported, a fact that may have important implications for the role of reelin during neuronal development in one of the few cerebral areas that maintains neuronal proliferation during adulthood.
We did not find evidence of reelin+ cells in the adult striatum in this work with the exception of the subventricular zone. Some groups have reported that reelin labeling decreases during early postnatal development up to undetectable levels at PND14 (Alcántara et al., 1998;Nishikawa, Goto, Hamasaki, Ogawa, & Ushio, 1999).
However, other groups reported the presence of reelin in the adult mouse striatum (Arlotta, Molyneaux, Jabaudon, Yoshida, & Macklis, 2008;Cocas et al., 2009;Sharaf, Rahhal, Spittau, & Roussa, 2015). It seems possible that the differences arise from discrepancies in the criteria to avoid false positive staining. It would be important to address this problem using more sensitive techniques.
Anyhow, it is interesting that the only place in the striatum where neurogenesis is maintained in adulthood, the subventricular zone, is where a clear and strong reelin labeling persists.

| Anterior commissure
White matter deterioration caused by perinatal asphyxia has been described elsewhere (Barkhuizen, van den Hove, et al., 2017;Bonestroo, Heijnen, Groenendaal, Van Bel, & Nijboer, 2015). A recent work showed a decrease in the thickness of the anterior commissure produced by PA in rabbits (Drobyshevsky, 2017). In this work we confirm this observation in rats and show that deep hypothermia reverses this alteration. This result may also have implications in schizophrenia since it has been reported the presence of white matter changes in schizophrenic patients (Bakhshi & Chance, 2015) including alterations in the anterior commissure (Choi et al., 2011). Inside the AC there were no variations in nucleus density (mostly belonging to glial cells).
The stain does not differentiate among oligodendrocytes and astrocytes.

| Limitations and methodological considerations
Rat development has several differences with humans. Among these, a major concern of some colleagues stems from the differences in the peak of speed in growth rate. With the intention of "solving" this problem, a few models were proposed where the asphyxia is produced in postnatal day 7. These models do not take into consideration a large number of factors that are specific of birth, such as the excitatory/inhibitory switch of GABAergic neurons, among many others.
We do not support the use of rats to PND7 for the study of perinatal asphyxia. We have already addressed this issue with more detail in previous works (see the discussion of Vázquez-Borsetti et al., 2018).
In Figure 1 Calretinin increase does not show significant differences in the nucleus accumbens, this is probably due to the combination of a low cell density with a small sampling area. This generates high error margins, but it is very probable the NAcc follows the same pattern as the dorsal striatum.
Of course, there could be alternative explanations for the reduction of calbindin+ neurons. PA could produce a disruption in the cellular expression of calbindin cells without killing them. If that were the case, we would observe an apparent decrease of neurons with calbindin, but the spiny neurons of the indirect pathway would be preserved. We find such explanation unlikely for many reasons. First, it is well known that PA kills cells and the mechanisms involved were widely described already (Northington, Chavez-Valdez, & Martin, 2011)

| Schizophrenia and dyskinesia
The striatum plays an important role in the physiopathology of schizophrenia and many authors consider that it is related to the symptomatic dimension involved in psychosis (Morrison & Murray, 2009).
Therefore, looking for strategies to avoid the damage of these neu- level, related to the ventral striatum (see (Haber, 2016). Interestingly, it has been reported that levodopa-induced dyskinesia is related to indirect pathway MSN excitotoxicity (Ivanova & Loonen, 2016).
Typical antipsychotic drugs are widely regarded to treat the positive symptoms of schizophrenia by antagonizing dopamine D 2 receptors expressed by striatal MSN of the indirect pathway (Sebel, Graves, Chan, & Surmeier, 2017). Antipsychotics act by antagonizing the D2 receptor that is inhibitory, which would generate an increase in the activity of the MSN of the indirect pathway. Our current hypothesis is that asphyxia during birth and antipsychotics act in opposite ways.
Asphyxia would decrease the density of MSNs that express the dopamine D2 receptor (inferred from the decrease of calbindin+ neurons by PA), while antipsychotics prevent these neurons from being inhibited by dopamine (see scheme in Figure 7).

| Final considerations
The factors that contribute to schizophrenia disease are multiple: social, neurodevelopmental and genetic. Each one may contribute to a spectrum of symptoms that eventually reach a certain level of severity that meets the requirements of the diagnosis of schizophrenia. The intention of this work is not to give a unique explanation of the origin of schizophrenia, we are not even sure that such explanation exists.
We propose a mechanism that can explain the generation of vulnerability to schizophrenia produced by PA. In previous works we had seen that deep hypothermia could prevent alterations that mimic negative symptoms of schizophrenia. Now we observed that the same treatment can reverse alterations linked to the positive symptoms or to the dimension of psychosis. Deep hypothermia can be a therapeutic alternative that prevents the deterioration generated by perinatal asphyxia such as dyskinesia and schizophrenia.

ACKNOWLEDGMENTS
We thank Francisco Urbano for his helpful insight and Luciana D'Alessio for her help with NeuN and calbindin immunohistochemistry. This research was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

CONFLICT OF INTEREST
The authors declare nonfinancial competing interests or other conflicts of interests for this article.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ORCID
Pablo Vázquez-Borsetti https://orcid.org/0000-0002-5974-5707 F I G U R E 7 It has been well described that medium spiny neurons comprise more than 95% of striatal neurons. All of them are GABAergic neurons expressing different profiles including calbindin that was reported in medium spiny neurons of the indirect pathway. PA decreases the number of calbindin neurons and increases other neuronal subpopulations. Deep hypothermia prevents these alterations. The present results suggest that therapeutic hypothermia could play an important role in the prevention of neurological and psychiatric pathologies related to alterations of medium spiny neurons such as dyskinesia and psychosis. Green circles represent the neuronal marker NeuN [Color figure can be viewed at wileyonlinelibrary.com]