Nesting Environment Provides Sex-Specific Neuroprotection in a Rat Model of Neonatal Hypoxic-Ischemic Injury

Abstract

Hypoxic-ischemic (HI) encephalopathy is a devastating injury that occurs when the fetal brain is deprived of oxygen and blood to a degree that may lead to neurological damage, seizing and cerebral palsy. In rodents, early environmental enrichment that promotes maternal care-taking behavior (mCTB) can improve neurobehavioral outcomes and protect against neurological decline. We hypothesized that an enhanced nesting environment would improve mCTB as measured by pup weight gain, and support greater HI recovery in developing rats. Pregnant dams (E15-16) were introduced to either control Standard Facility (SF) housing or closed nestbox (CN) conditions and maintained in larger cages through pup weaning. On postnatal day (PND) 7, male and female Long-Evans rat pups (N = 73) were randomly sorted into one of two surgical conditions: control and HI. HI pups received isoflurane anesthesia and right carotid artery ligation, a 2-h rest followed by 90 min exposure to a moist hypoxic (92% N, 8% O2) chamber. Pups (PND 8) were weighed daily, and tested on the Morris Water Maze (MWM) task (PND 35-50). Results demonstrate significant differences afforded to male and female pups based on weight measure, where CN-rearing modifies pre-weaning adolescent weights in females and increases post-weaning weights in males and females by an average of 10 g. Following successful MWM training and acquisition (PND 35-37), both male and female CN-raised animals demonstrated faster latency to find the hidden platform (HP) during HP trials (PND 38-42) and appeared to freely explore the MWM pool during an additional probe trial (PND 43). Moreover, after sacrifice (PND 60), CN rearing created sex-specific alterations in brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF) immunopositive cell staining of the dorsomedial striatum and CA1 of the hippocampus. CN-rearing afforded HI males higher BDNF levels in the striatum and produced greater GDNF levels in the hippocampus of HI-injured females. These results suggest that early life environmental enrichment positively modifies nesting environment, increases weight gain, as well as spatial learning and memory in a sex-specific directionality. Our findings also implicate correlative changes in corticolimbic neurotrophin protein levels in the CN-reared animals that may contribute to these benefits.

Keywords: Long Evans rats; Morris water maze; Rice-Vanucci P7 HI model; environmental enrichment; hippocampus; neonatal hypoxic ischemic injury; neurogenesis.

Figure 1

Picture of Closed Nestbox (CN) inside standard cage with bedding. CN was added to the dam’s cage at embryonic day (ED) 10 (E10-15) and remained until weaning on postnatal day (PND) 21.

Figure 2

Depiction of study timeline indicating the introduction of the CN enrichment at embryonic day 10–15, surgery on PND 7, Morris Water Maze (MWM) testing and study termination.

Figure 3

Nissl-stained coronal hippocampal sections (ipsilateral to injury) from representative males exposed to Hypoxic-ischemic (HI) injury and reared in CN (top panel) and Standard Facility (SF; bottom). CN rearing provided enduring neuroprotection in late adolescence following neonatal HI insult. Scale bar = 200 μm for all images (bottom right); 20× magnification.

Figure 4

(A,B) Mean latency by study condition in visible platform (VP) training. The following graphs mark the average latency of animals to reach the platform over the course of the four water maze trials each day for three consecutive days. The lines compare average latency for animals in the CN and SF housing conditions according to surgical condition: (A) Control, (B) HI Injury. Only main effects were observed for latency for males (F₍₂₎ = 103.20, p < 0.05), and females (F₍₂₎ = 71.30, p < 0.05) with both demonstrating decreased latency across the three training days indicating successful task acquisition.

Figure 5

(A,B) Mean latency by surgical and housing condition in hidden platform (HP) testing for males and females. The following graphs mark the average latency of animals to reach the HP over the course of the four trials each day for five consecutive days. The lines compare average latency for animals in the CN and SF housing conditions according to surgical condition: (A) Control, (B) HI Injury. There were significant differences between surgical conditions regardless of housing condition for males (F₍₁₎ = 11.35 p < 0.001; HI males relative to Control males). Females demonstrated a significant interaction effect between surgical condition and housing condition (F₍₁₎ = 9.37 p < 0.001; HI SF as compared to all other groups, same sex), with HI SF females performing significantly worse than all other groups.

Figure 6

(A,B) Probe trial: average swim path by surgery condition and housing for males and females. The following heat maps mark the average swim path during the probe trial. The markings represent aggregate data from CN and SF housing conditions for each sex according to surgical condition: (A) Control, (B) HI Injury. The maps illustrate that HI SF animals had significantly longer swim paths than either HI CN, Control SF, or Control CN animals (F₍₁₎ = 7.115, p < 0.01). For swim speed velocity, there was no difference between surgical conditions, but there was a significant effect of housing condition, with SF females demonstrating a faster swim speed than CN females (F₍₁₎ = 7.05, p < 0.01).

Figure 7

Immunoflourescent staining for neurotrophic factors and DAPI vary by surgical and housing conditions in males. (A) Ipsilateral hippocampal CA3 staining for BDNF in HI-injured males housed in CN (left panel) and SF (right panel). (B) GDNF-positive cells in males that suffered HI insult and were reared in CN (left panel) and SF (right panel). (C) DAPI-stained cells of male rats following HI insult and CN (left panel) and SF (right panel) pre-weaning environments. Scale bar in bottom right image = 500 μm, 20× magnification for all images.

Figure 8

HI injury differentially affects neurotrophic factors BDNF, GDNF and DAPI in the hippocampus of male rats reared in CN and SF conditions. (A) BDNF-positive staining in CA3 region of hippocampi of male rats after HI injury and CN (left panel) and SF (right panel) rearing. (B) Anti-GDNF positive cells in the hippocampus of HI-injured male rats from CN (left panel) and SF (right panel) housing. (C) Staining of DAPI cells in HI males from CN (left panel) and SF (right panel) environments. Scale bar = 500 μm, 20× magnification for all images.

Figure 9

(A–F) Immunopositive cell counts for GDNF, DAPI and BDNF in hippocampus by surgery and housing conditions for both sexes. Bar graphs show average ±SEM counts for (A–B) GDNF-positive cells, (C–D) DAPI-positive cells and (E–F) BDNF-positive cells in the hippocampus. No significant main effects or interactions were observed for GDNF. For DAPI, male control rats reared in CN had fewer immunopositve cells relative to all other groups, ***p < 0.001. HI injury males reared in SF had fewer cells relative to Control males reared in the same condition, *p < 0.05, **p < 0.01.

Figure 10

(A–F) Positively stained cells for BDNF GDNF, DAPI in the striatum by surgery condition and rearing in males and females. (A-B) In both males and females, CN rearing increased GDNF counts following HI injury, *p < 0.05, relative to Control CN; ⁺p < 0.05, compared to HI injury SF rearing. (C-D) No differences were observed for DAPI staining for males or females for any of the surgical or housing conditions. (E) CN rearing improved BDNF stained cells in the striatum of males, regardless of injury (control and HI); **p < 0.01, CN vs. SF, same injury group. The overall number for BDNF-positive cells was greater in the control males relative to HI injured males, ***p < 0.001.