Calbindin-1 Expression in the Hippocampus following Neonatal Hypoxia-Ischemia and Therapeutic Hypothermia and Deficits in Spatial Memory

Hypoxia-ischemia (HI) is one of the most common causes of perinatal brain injury. Complete or partial disruption of blood flow to the brain impairs normal brain function by reducing the delivery of oxygen and glucose necessary for aerobic metabolism [1-3]. Along with the basal ganglia and the cortex, the hippocampus is one of the regions with the highest metabolic demands and vulnerability to HI [3, 4]. Harmful increases in intracellular Ca²⁺ concentrations lead to mitochondrial failure and neuronal death [2, 4, 5]. Ca²⁺ dysregulation may persist long after the initial HI insult and leads to neuroinflammation and ongoing injury [6].

Neurons use EF-hand type Ca²⁺-binding proteins (CBPs) to buffer out excess calcium and achieve homeostasis after an insult [2, 7, 8]. These CBPs, including calbindin-1 (Calb1), are also involved in many neuronal functions, such as synaptic transmission, cell signaling, the release of neuropeptides, gene expression and activation, and cell death [7, 9-12]. Inhibition of Ca²⁺ influx attenuates neuronal cell damage from ischemic injury [8]. Thus, the relative expression of CBPs, or the lack thereof, plays an important role in neuronal injury in response to an insult such as HI. Although these proteins often work in conjunction with each other, there are distinct differences in their basic functions. Specifically, Calb1, mainly expressed by GABAergic interneurons of the cornus ammonis (CA) subfields of the hippocampus and the granular cells of the dentate gyrus (DG) [7, 11], has the highest affinity to buffer calcium [11], and has been linked to the preservation of hippocampal-dependent memory in the mouse [11, 12].

Neonatal HI injures the basal ganglia-thalamus and cortical tracks and results in motor impairments, which are attenuated by therapeutic hypothermia (TH) [13-16]. However, a significant proportion of survivors of neonatal HI brain injury suffer from cognitive and memory impairments, suggesting that TH does not fully protect the hippocampus, an essential brain region for the consolidation of memory [7, 12, 13, 16]. Persistent disturbance in Calb1 expression within the hippocampus may play a critical role in memory outcomes following neonatal HI injury. There is a segregation of the rodent hippocampus along the dorsoventral axis, with the dorsal hippocampus (anterior in the mouse) functionally linked to spatial learning and memory [17], so the study of the relationship between Calb1 and memory outcomes requires stratification according to hippocampal section in the anteroposterior axis. Here, we aimed to study the expression of hippocampal Calb1 following HI and TH, and the link with the degree of astrogliosis and memory outcomes in a mouse model. We hypothesize that, despite TH, neonatal HI persistently impairs Calb1 expression in the hippocampus, a change linked to memory deficits in the mouse.

Male and female C57BL6 (Charles River Laboratories, Wilmington, MA, USA) pups were used for these experiments, totaling 215 mice. All efforts were made to minimize the number of mice used and their suffering.

Neonatal HI brain injury of postnatal day 10 (P10) mice was induced using the Rice-Vannucci model adapted for neonatal mice as previously published. In short, the mice were anesthetized with an inhaled isoflurane (3% for induction and 1% for maintenance) mixture with nitrous oxide prior to the induction of brain HI by unilateral right carotid artery ligation. They were returned to their cages for a 1-h resting period followed by 45 min of hypoxia at FiO₂ = 0.08 in an acrylic glass chamber. After HI, mice were randomized to normothermia (NT) at 36°C or TH at 31°C for 4 h, and then returned to their cages. Surgery lasted 3–5 min. Mice randomly assigned to the sham group received an inhaled isoflurane/nitrous oxide mixture for 5 min at similar concentrations as described above for the NT and TH groups and then returned to their cages. This mouse model of neonatal HI and TH has been validated by T2 MRI techniques, demonstrating sustained neuroprotection against cerebral and cortical atrophy at 8 and 20 days after HI injury at P10 and a lack of sustained protection of the hippocampus [18]. This differential regional protection afforded by TH in this model resulted in protection of the motor domains and a lack of protection of the memory domains of neurodevelopment [19]. After exclusions, we studied the brains in 3 different cohorts of animals at P11 (sham n = 9; NT n = 10; TH n = 11), P18 (sham n = 9; NT n = 7; TH n = 14), and P40 (sham n = 37; NT n = 31; TH n = 35) on immunohistochemistry (IHC). Mice were killed via exposure to 20% (v/v) mixture of isoflurane in propylene glycol by the 1-drop exposure method [20] prior to being exsanguinated with 0.1 M cold PBS (pH 7.4) via cardiac perfusion in the left ventricle. Brains were perfused with 4% paraformaldehyde (PFA) at 4 mL/h and postfixed overnight on 4% PFA prior to being cryoprotected in 30% sucrose in PBS until they sank. All brains were then frozen and stored at –80°C prior to being cut on a freezing microtome at 50 µm.

The use of the term “anterior” or “posterior” reflects the position of the coronal brain section in the anteroposterior axis. For all experiments, we evaluated the hippocampal sections located within the mouse brain. Sections were classified as anterior if the fasciola cinereum was adjacent to the dorsal third ventricle and before the emergence of the dorsal subiculum. They were classified as posterior if the CA3 subfield crossed the level fasciculus retroflexus but was positioned prior to the emergence of the DG granular cell layer in the ventral portion of the coronal section [9]. Based on the description above, the hippocampus located in our anterior coronal brain sections corresponds to the dorsal/septal hippocampus, while that located in our posterior coronal brain sections corresponds to the transition between the dorsal/septal and ventral/temporal hippocampus. Thus, the hippocampus in the posterior coronal sections of the brain does not correspond exclusively to the ventral hippocampus, but shows an arrangement of the dorsal and ventral hippocampus in the upper and lower areas, respectively.

The hippocampus within the anterior and posterior coronal brain sections was assessed for severity of injury using Nissl staining and glial fibrillary acidic protein (GFAP) scoring [9]. Evidence of early (P11) cell death with nuclear changes characterized by pyknosis, karyorrhexis, and karyolysis, apoptotic cell bodies, and delayed (at P18 and P40) columnar injury of the CA1 and CA3 subfields without obliteration on Nissl staining were included in the analysis. GFAP scores of 3–7 per subfield or 9–21 in the whole-hippocampus analysis on IHC suggested injury/astrogliosis and were included in the analysis [9]. Pups demonstrating either minimal or no injury (2 at P11, 4 at P18, and 5 at P40) or an obliterated hippocampus (2 at P11, 4 at P18, and 9 at P40) were not included in the analysis.

For IHC, tissue was fixed with 4% PFA and cryoprotected with a 15/30% sucrose gradient as previously described prior to storage at –80°C, and then cut at 50 µm with a freezing microtome [21]. Floating IHC was performed as previously described [21] with whole-rabbit antisera anti-Calb1 (Cell Signaling Technology, Inc, Beverly, MA; 1: 250), or anti-GFAP (DAKO/Agilent Technologies, Santa Clara, CA, USA; 1: 1,000) followed by goat anti-rabbit antibody (1: 200) used as the secondary antibody and DAB. Cresyl-violet (CV) staining was performed to assess injury at P11, P18, and P40 and for volumetric analysis.

Calb1 (CS13176; RRID:AB_2687400): rabbit monoclonal antibody raised against a recombinant protein specific to the N-terminus of whole Calb1 protein of human origin detecting a product at 28 kDa (2 μg/mL). GFAP (DAKO Z0334; RRID:AB_10013382): rabbit polyclonal antibody raised against GFAP isolated from cow spinal cord with no reported cross reactivity (1 μg/mL).

Calb1 IHC and CV-stained slides were imaged using a light microscope (Nikon Eclipse E400, Nikon), to produce high-resolution photomicrographs, and then analyzed using ImageJ v1.8.0 software (NIH, Bethesda, MD, USA) (Rasband 1997–2018).

Using the free draw tool, the whole hippocampus and the DG field were outlined separately. All images were converted to 8-bit images and globally calibrated to optical density (OD). The background was removed from the images to measure the Calb1 staining. The background was obtained by drawing 10 circles free of Calb1 staining, 5 in the lacunosum molecular layer and 5 in the oriens layer. The average OD score from these 10 background circles was subtracted from the whole-hippocampus average OD score to create the adjusted mean OD for the hippocampus. The mean OD of the CA field was determined by the following formula: CA mean OD = [(mean hippocampal OD•hip area) – (mean DG OD•DG area)]/(hippocampal area – DG area). Thus, the Calb1 OD used for the analysis and correlations was the average of the OD measured in every pixel contained within the region of interest (whole hippocampus, CA field, or DG).

We calculated residual volumes using the hippocampal areas (mm²) obtained from 5 representative 50-µm, CV-stained coronal brain sections positioned 600 µm apart in the anteroposterior axis of the brain at P40. Total residual volume was extrapolated using the following formula:

Hippocampal volume (mm³) = ∑_{i = 1 (n = 5)} [S_i × 0.05] + ∑_{i = 1 (n = 4)} [(S_i + S_i+1) × 0.6/2]

where S = area of hippocampal section in mm², i = section position in the anteroposterior axis, and n = number of sum repeats.

The P40 cohort underwent behavioral testing (Y-maze) between P22 and P26 before harvesting the brain at P40. Testing was conducted in a noise-free room between 1 and 6 p.m. The order of testing the mice was randomized for each test within the litters. Each litter had a maximum of 6 pups, with up to 2 litters during each behavioral session. Pups were habituated in the experimental room for 30 min prior to the start of testing. All scored tasks were recorded using videos which were reviewed and scored manually by 2 team members blinded to the treatment groups (JGH and DS), and electronically using AnyMaze v5.1 (Stoelting Co. Wood Dale, IL, USA). The mice were returned to their cages following testing.

The Y-maze test was used to assess spatial working memory in rodents. Uninjured mice will choose to investigate a new arm before returning to one recently visited. The apparatus was made of acrylic glass forming a “Y” with 3 identical arms positioned at 120° angles. There are 2 phases each lasting 5 min: phase 1 to assess working memory and phase 2 to assess spatial and recognition memory. During both phases, the mice started the test in the center of the maze. During phase 1, the sequence of arm entries was recorded. Spontaneous alterations performance (SAP) assesses working memory as the mice visit 3 consecutive different arms of the maze. Impaired working memory is determined by higher alternate-arm returns (AAR), i.e., 3 consecutive arm visits in which the first and third arm visit is the same, and same-arm returns (SAR), i.e., 2 successive visits to the same arm. Phase 2 was performed 3 days after phase 1. During the prerecorded phase, mice explore the maze with 1 arm blocked (the novel arm) for 5 min. After a 30-min resting period, all arms were open for the mice to explore the maze once more for 5 min. The percentage of time spent and the number of entries into the novel arm were analyzed.

Along with CV staining, GFAP IHC was used to assess the degree of astrogliosis as a surrogate hippocampal injury in order to exclude those mice demonstrating minimal or no injury. We used a semiquantitative GFAP-derived scoring system developed in our laboratory [9] for this purpose and also to establish the correlation between persistent astrogliosis and changes in Calb1 expression. This scoring system takes into account the dispersion of hippocampal immunoreactivity and the morphological characteristics of astrocytes [22, 23] in the CA1 and CA3 subfields and in the DG in the hippocampus positioned in the anterior and posterior coronal brain sections. Scoring was based on the abundance of GFAP staining and glial scars, astrocyte body size, the number and thickness of the branches, and the overlap of astrocytic domains at a higher magnification [9]. Representative high-magnification photomicrographs showing GFAP-stained astrocytes within the hippocampal CA1 subfield at P40 can be seen in online suppl. Fig. 1 (see www.karger.com/doi/10.1159/000497056 for all online suppl. material). The presence of astrocytes with small somas and few or many thin branches without overlapping domains was granted 1 or 2 points for the subfield, respectively (online suppl. Fig. 1A₁, A₂). The identification of astrocytes with large somas and many thick branches with overlapping domains was granted 3 points (online Suppl. Fig. 1A₃), while the presence of astrocytes with even larger soma, forming 1, 2, or ≥3 glial scars, was granted 4, 5, or 6 points, respectively (online suppl. Fig. 1A₄). Lastly, diffuse astrocytic activation through the subfield was granted 7 points. The cumulative score was the sum of the scores for each of the 3 subfields (CA1, CA3, and DG), which varied from 1 to 7. This resulted in a cumulative score of 3–21 for the whole hippocampus and 2–14 for CA field.

Multiple groups were analyzed using nonparametric Kruskal-Wallis (KW) H one-way ANOVA stratified by sex, with post hoc pair analysis using the Dunn-Bonferroni test. All results were presented as box and whisker plots, where the box was limited by the 25th and 75th percentiles and the solid line represented the median. Significance was assigned by p ≤ 0.05 adjusted for multiple comparisons in all cases. Nonparametric Spearman Rho correlations were applied between Calb1 expression, hippocampal residual volume, GFAP score, and memory outcome. Analysis was performed using SPSS v24v (IBM Corp., Armonk, NY, USA).

In the hippocampal sections located within both the anterior and posterior coronal sections, Calb1 expression increased between P11 and P40. In the hippocampus within the anterior coronal sections, Calb1 expression increased from 0.167 arbitrary units (a.u.) (IQR 1.157–0.177) at P11 to 0.204 a.u. (0.161–0.263) at P18, and 0.334 a.u. (0.277–0.339) at P40 (Fig. 1A₁–A₄). The expression in the CA field within the anterior coronal sections was, on average, 0.105 a.u. (0.103–0.110) at P11, 0.117 a.u. (0.936–0.147) at P18, and 0.284 a.u. (0.196–0.367) at P40 (H 22.7; df 2; KW p < 0.001; P11 vs. P40 and P18 vs. P40 adjusted p = 0.001; Fig. 1A₅). Thus, in the anterior brain sections, the 2-fold increase in hippocampal Calb1 expression between P11 and P40 (H 19.9; df 2; KW p < 0.001; P11 vs. P40 adjusted p = 0.001) was specifically linked to the increase within the CA field (Fig. 1A₅), but not the DG (Fig. 1A₆). Similarly, in the posterior brain sections, hippocampal Calb1 expression increased from 0.239 a.u. (0.194–0.352) at P11 to 0.415 a.u. (0.362–0.445) at P40 (H 20.4; df 2; KW p < 0.001; P11 vs. P40 adjusted p = 0.002; Fig. 1B₁–B₄). The 75% increase in the hippocampal Calb1 expression between P11 and P40 was dependent on the increase within the CA field of the posterior brain sections (H 12.4; df 2; KW p = 0.002; P11 vs. P40 adjusted p = 0.003; Fig. 1B₅) but not the DG (Fig. 1B₆). Collectively, the expression of hippocampal Calb1 increased by 54 and 96% between P11 and P18 (H 29.2; df 2; KW p < 0.001; adjusted p < 0.001) and P18 and P40 (adjusted p < 0.001), respectively. This changed was linked to the increase in the CA field (H 27.2; df 2; KW p < 0.001; P11 vs. P18 and P18 vs. P40 adjusted p < 0.001; data not shown).

Neonatal HI did not alter the developmental increase in hippocampal Calb1 expression between P11 and P18 in the anterior coronal brain sections (Fig. 2A₁–A₃). However, deficits in expression of Calb1 were documented in the hippocampus as early as 8 days after injury (P18, Fig. 2B) in the posterior brain sections. Specifically, the expression of Calb1 within the CA field was 18% lower 8 days after HI injury (H 7.1; df 2; KW p = 0.02; adjusted p = 0.05 vs. sham) and TH did not provide protection (adjusted p = 0.02 vs. sham; Fig. 2B₂). Impairment in the expression of Calb1 in the hippocampus progressed by P40 (30 days after injury) in the posterior brain sections. At P40, hippocampal Calb1 expression in the posterior brain sections of injured mice was 20% lower than in shams (H 22.4; df 2; KW p < 0.001; adjusted p ≤ 0.001 vs. sham) and TH did not prevent this deficit (–24%; adjusted p = 0.002 vs. sham; Fig. 3A₁). The developmental increase in Calb1 expression in the CA field between P11 and P40 (Fig. 1B₅) was impaired by neonatal HI injury in the posterior brain sections (–25%, H 10.8; df 2; KW p = 0.005; adjusted p = 0.03 vs. sham; Fig. 3A₂). Even the mice treated with TH after HI injury had 32% lower Calb1 expression in the CA field compared to shams (p = 0.008; Fig. 3A₂). Again, no differences in Calb1 expression were seen in the DG at P40 (Fig. 3A₃). Calb1 deficits in the hippocampus and CA field were similar in male and female mice in the posterior brain sections (online suppl. Fig. 2).

Our previously published GFAP-derived scoring system [9] was applied to grade astrogliosis and correlate with Calb1 expression at P40. Representative photomicrographs show GFAP staining of the hippocampus within the anterior (online suppl. Fig. 1B) and posterior (Fig. 4) coronal brain sections at P11, P18, and P40. The median (IQR) GFAP scores of sham, HI/NT, and HI/TH mice in the hippocampus in the anterior brain sections were 3 (3–4), 16 (11–20), and 12 (10–16) (H 68.7; df 2; KW p < 0.001), respectively; in the posterior brain sections they were 3 (3–4), 14 (10–21), and 12 (10–15) (H 71.5; df 2; KW p < 0.001), respectively. Median GFAP scores of sham, HI/NT, and HI/TH mice in the CA field in the anterior sections were 2 (2–3), 12 (8–14), and 9 (7–12) (H 71.6; df 2; KW p < 0.001), respectively; in the posterior sections they were 2 (2–3), 11 (7–14), and 9 (8–11) (H 71.4; df 2; KW p < 0.001), respectively. The GFAP scores in the hippocampus (Spearman Rho p < 0.001, r = –0.47; Fig. 3B₁) and the CA subfield (Spearman Rho p < 0.001, r = –0.39; Fig. 3B₂) from the posterior brain sections inversely correlated with their respective Calb1 expressions at P40. There was no correlation between GFAP score and Calb1 expression in the DG of the posterior sections (Fig. 3B₂).

The percentage SAP in Y-maze phase 1 was lower in HI-injured males by 40% (H 28.9, df 2, KW p < 0.001, adjusted p < 0.001) and females by 38% (H 19.3, df 2, KW p < 0.001, adjusted p < 0.001) when compared to shams (Fig. 5A). Accordingly, the percentage AAR was 2.6-fold higher in HI-injured males (H 22.6; df 2; KW p < 0.001; adjusted p < 0.001) and 1.9-fold higher in females (H 23.5; df 2; KW p < 0.001; adjusted p = 0.001) when compared to shams (data not shown). This resulted in 2.2- and 2-fold higher total impairment after HI injury in males and female HI-injured mice, respectively (KW p < 0.001 for both; Fig. 5B) versus shams. The time spent exploring the new arm during phase 2 of the Y-maze was approximately 50% less in male (KW p = 0.002; adjusted p = 0.03) and female (KW p = 0.02; adjusted p = 0.05) injured mice versus shams (Fig. 5C). TH did not attenuate any of the deficits documented during the Y-maze testing. In general, Calb1 expression in the whole hippocampus (Fig. 5A₁–C₁), and specifically in the CA field (Fig. 5A₂–C₂) within the posterior coronal brain sections, directly correlated with percentage SAP (Fig. 5A₁, A₂) and time spent exploring the new arm (Fig. 5C₁, C₂) and inversely correlated with the percentage of total impairment (Fig. 5B₁, B₂). However, the link between Calb1 expression and the performance in the Y-maze was more robust in females than in males. In female mice only, Calb1 expression in the CA field of the posterior brain sections directly correlated with the percentage SAP and time spent exploring the new arm (Fig. 5A₂, C₂). There were no significant correlations between hippocampal Calb1 expression in the anterior brain sections and Y-maze performance.

Neonatal HI produced a 32% (H 17.5; df 2; KW p < 0.001; adjusted p < 0.001 vs. sham) and 25% (H 16.7; df 2; KW p < 0.001; adjusted p = 0.006 vs. sham) decrease in the hippocampal volume at P40 in male and female mice, respectively. TH did not mitigate hippocampal atrophy (adjusted p < 0.001 [males] and 0.001 [females] vs. sham; Fig. 6A). Residual hippocampal volumes did not correlate with hippocampal Calb1 mean OD at P40 (Fig. 6B). Nevertheless, residual hippocampal volumes predicted memory outcomes using Y-maze testing. Hippocampal volume was directly correlated with percent SAP in Y-maze phase 1 (r = 0.45, p = 0.02 [males]; r = 0.47, p = 0.01 [females]), percentage of time spent exploring the new arm during Y-maze phase 2 (r = 0.39, p = 0.03 [females]) and was inversely correlated with the percentage of total impairment (r = –0.42, p = 0.03 [males]; r = –0.44, p = 0.02 [females]).

We show that hippocampal Calb1 expression developmentally increases between P11 and P40, specifically within the CA subfields, but not in the DG in the anteroposterior axis of the brain. Neonatal HI produces delayed and late deficits in Calb1 expression exclusively within the CA field of the hippocampus in the posterior sections, but not in the anterior sections, of the mouse brain. These persistent deficits in hippocampal Calb1 expression after neonatal HI in the posterior sections of the brain are: (i) similar in both sexes, (ii) inversely correlated with the degree of astrogliosis, (iii) not correlated with hippocampal atrophy, and (iv) not attenuated by TH. Although neonatal HI produced deficits in spatial working memory in the Y-maze in both male and female mice, a direct correlation of deficits in the expression of Calb1 in the CA subfields is only documented in the posterior brain sections and more robustly in the female mice at P40. To our knowledge, this is the first report documenting the persistent deficits in expression of the Ca²⁺-buffering protein Calb1 after neonatal HI, the lack of response to TH, and a correlation with memory deficits.

The developmental increase in hippocampal expression of Calb1 throughout the anteroposterior axis of the brain was specifically linked to the increase within the CA subfields. Our findings are in agreement with the developmental increase of Calb1 described between P6 and P35 in the rat hippocampus [24], and between P7 and P22 in other regions of the brain [25]. The increase in Calb1, and other CBPs, is mostly linked to the postnatal maturation of interneurons within the CA subfields and of the mossy fiber system between the DG and CA3 [9, 26-31]. Calb1 plays an important role in protecting the brain against stroke, traumatic brain injury [32], neurodegenerative disorders [33-37], and aging [38]. In the developing brain, massive shifts in intracellular Ca²⁺ produced by perinatal brain injury or kainic acid administration induce significant hippocampal injury that is attenuated by the upregulation of Calb1 expression [39]. Similarly, study of the aging human brain suggests that subtle changes in Ca²⁺ homeostasis produced by a decrease in CBPs lead to drastic deficits in cognitive function and increases vulnerability to neurodegeneration [40].

Behavioral, functional, anatomical, and connectivity analysis support the segregation of the hippocampus into anterior, middle, and posterior sections in primates, and dorsal, intermediate, and ventral in rodents [17, 41-45]. While the dorsal hippocampus (positioned anteriorly in rodents and corresponding to the posterior hippocampus in primates) is primarily involved in cognitive function and the retrieval of memories [41, 46, 47], the ventral hippocampus (mostly positioned posteriorly in rodents and corresponding to the anterior hippocampus in primates) is involved in anxiety and emotions [41]. The function of the intermediate hippocampus is not well defined, but it appears to overlap the functions of the dorsal and ventral hippocampus and also plays a role in spatial learning and memory [41, 48].

Identification of the boundaries of the dorsal, intermediate, and ventral hippocampus in coronal sections of the mouse brain, as in our experiments, is challenging. Based on the anatomical limits used to classify the anterior and posterior coronal brain sections that we used (Methods section: “Terminology”), we consider that our anterior brain sections indisputably contained sections of the dorsal hippocampus, while our posterior brain sections contained a combination of the dorsal and intermediate hippocampus, and ventral hippocampus to a lesser extent, transitioning from the top to bottom of the section, as described by Fanselow and Dong [41]. Here, we show that neonatal HI produces deficits in Calb1 exclusively in the CA subfields of the hippocampus contained in the posterior coronal sections but not in the anterior sections of the brain.

In our model, the CA subfields were more selectively vulnerable to HI injury than the DG [49], but there is no obvious explanation for the differential expression of Calb1 between hippocampal sections closer to the septal pole (anterior brain sections) and those positioned intermediately between the septal and temporal poles (posterior brain sections). Our analysis of astrogliosis as a surrogate of injury did not suggest differential injury between the hippocampal CA subfields in the anterior and posterior coronal sections (a GFAP score of 12 and 11, respectively). However, given that the developmental increase in Calb1 in the CA subfields in the anterior brain sections occurred after P18 (Fig. 1) and that in the posterior brain sections occurred before P18, HI injury at P10 most likely altered the maturational mechanisms leading to Calb1 expression occurring closer to the insult, and so predominantly affected the hippocampus in the posterior coronal brain sections.

Thirty days after HI, Calb1 deficit within the CA subfields contained in the posterior brain sections directly correlated with worse spatial memory outcomes. This link appears to be more robust in female mice than in male mice and is not dependent on the degree of hippocampal atrophy resulting from HI injury. Although functionally spatial working memory has been linked to the ventral hippocampus as well [48, 50], the strongest evidence [41] suggests that the dorsal, and likely the intermediate, hippocampus (contained within the posterior coronal brain sections) would have the strongest influence on the Y-maze outcomes. However, the lack of correlation between Calb1 expression in the dorsal hippocampal sections (contained within the anterior brain sections) and the memory outcomes in our experiments was puzzling. We speculate that the limited variability in Calb1 OD due to the lack of effect of the HI insult compared to in sham mice in the most dorsal portion of the hippocampus did not allow the for a directional correlation with memory outcomes. Alternatively, the intermediate hippocampus may have a more important role in memory outcomes than previously thought.

Decrease in Calb1- and parvalbumin-expressing interneurons in the CA subfields of the mouse hippocampus 8 days after neonatal HI was reported recently by our group [9]. Calb1-positive interneurons are essential for cognition and memory preservation [51-54]. The GA-BAergic control of pyramidal cells by hippocampal interneurons allows the preservation of mechanisms of long-term plasticity, a molecular proxy of memory, and so a GABAergic deficit leads to memory impairments [55]. Evidence between changes in Calb1 and memory outcomes has been extensively described in traumatic brain injury [56], hippocampal sclerosis [57], and Alzheimer’s disease [58]. Any reduction in Calb1 expression appears to be critical enough to impair the ability of the hippocampus to function normally [59]. Our results led us to speculate that the loss of Calb1 immunoreactivity is, at least in part, linked to the decrease of Calb1-positive interneurons in the postnatal CA subfields contained in the posterior murine brain. To our knowledge, this is the first report providing evidence that, similar to many other neuropsychiatric and neurodegenerative disorders, neonatal HI may impair Calb1 expression, in this case by preventing normal development.

We evaluated astrogliosis using the semiquantitative GFAP-derived scoring system recently developed in our laboratory [9]. The severity of astrogliosis as determined by this scoring system, inversely correlated with Calb1 expression in the CA subfields of the posterior brain sections. We were unable to determine whether persistent astrogliosis led directly to impaired Calb1 expression in the CA subfields by 30 days after HI, but it appears that the severity of HI injury, as suggested by the degree of hippocampal atrophy, does not fully explain the Calb1 deficits. Although associations between Calb1 loss and astrogliosis in the hippocampus have been previously described following kainic acid injury [60], ethanol exposure [61], viral infections [62], and even exposure to radiofrequency [63], the causal mechanism for these 2 events is still missing and further study is required.

We acknowledge several limitations to our study. We evaluated the hippocampal sections contained within the anterior and posterior coronal sections of the mouse brain. This enabled us to study the dorsal hippocampus closest to the septal pole, but not the intermediate or ventral hippocampus individually, as the hippocampal sections contained in the posterior coronal section are a combination of all of these (as previously published [41]). Thus, the role of the isolated intermediate hippocampus in memory outcomes requires further investigation in association with Calb1 deficits. Additionally, our neonatal HI model produced unilateral injury, and thus compensation from the contralateral (hypoxia-exposed) hemisphere may have attenuated the behavioral outcomes. Despite these limitations, our laboratory has extensive experience in behavioral testing using this mouse model and has obtained consistent results demonstrating impaired memory outcomes (using Y-maze testing) and a lack of response to TH [18, 19]. We did not analyze the electrophysiological properties of Calb1-positive interneurons within the CA subfields to assess function and alterations in the mechanisms of long-term synaptic plasticity to link Calb1 deficit to memory deficits. Lastly, we acknowledge that we only included mice without an obliterated hippocampus due to technical limitations when processing the samples and because HI injury almost fully eliminated the CA subfields from the analysis.

In conclusion, neonatal HI produces a long-lasting impairment in the increase of Calb1 in the CA subfields of the hippocampus during a critical stage of development, and TH does not attenuate the deficit. Late Calb1 deficit correlates with prolonged astrogliosis. Further studies are needed to understand the mechanistic link between deficits in spatial memory and Calb1 expression and the functional role of the intermediate rodent hippocampus.

The authors thank the editorial input from Dr. Frances Northington, the technical support of Mr. Charles Lechner, Armand Sandjeu and Ms. Elizabeth Krisanda and the administrative support of Mrs. Rosie Silva.

The experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine and carried out with standards of care and housing in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services 85–23, 2011.

We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

The experiments and investigators were funded in parts by grants from the National Institute of Neurological Disorders and Stroke (KO8NS096115 – R.C-V.), the Johns Hopkins University-School of Medicine Clinician Scientist Award (R.C-V.), the Sutland-Pakula Endowment for Neonatal Research (R.C-V.), and by the Doctoral Diversity Program funded by the Health Resource Services Administration (J.G.H).

J.G.-H. and R.C.-V.: experimental design, imaging and analysis, and initial draft preparation; J.G.-H., D.S., and J.D.: behavioral testing; J.G.-H. and D.F.: immunohistochemistry; R.C.-V.: statistical analysis; and all authors: critical reviews of the manuscript and approval of the final version.