Hypoxia-Ischemia-Mediated Effects on Neurodevelopmentally-Regulated Cold-Shock Proteins in Neonatal Mice Under Strict Temperature Control

Abstract

Background:

Neonates have high levels of cold-shock proteins (CSPs) in the normothermic brain for a limited period following birth. Hypoxic-ischemic (HI) insults in term infants produce neonatal encephalopathy (NE), and it remains unclear if HI-induced pathology alters baseline CSP expression in the normothermic brain.

Methods:

Here we established a version of the Rice-Vannucci model in PND 10 mice that incorporates rigorous temperature control.

Results:

Common carotid artery (CCA)-ligation plus 25 min hypoxia (8% O₂) in pups with targeted normothermia resulted in classic histopathological changes including increased hippocampal degeneration, astrogliosis, microgliosis, white matter changes, and cell signaling perturbations. Serial assessment of cortical, thalamic, and hippocampal RNA-binding motif 3 (RBM3), cold-inducible RNA binding protein (CIRBP), and reticulon-3 (RTN3) revealed a rapid age-dependent decrease in levels in sham and injured pups. CSPs were minimally affected by HI and the age point of lowest expression (PND 18) coincided with the timing at which heat-generating mechanisms mature in mice.

Conclusion:

The findings suggest the need to determine if optimized therapeutic hypothermia (depth and duration) can prevent the age-related decline in neuroprotective CSPs like RBM3 in the brain, and improve outcome during critical phases of secondary injury and recovery after NE.

Introduction

Mammalian cold-shock proteins (CSPs) are critical mediators of the cellular adaptive response to cold stress^1,2. While the translational efficiencies of most proteins are coupled to the activity of temperature-sensitive enzymes involved in cap-dependent synthesis, CSPs evolved unique molecular mechanisms allowing them to escape translational repression at cooler temperatures^3–5. Together they modulate critical biological processes including inflammation^6–8, fever⁹, neuroprotection^5,10–12, neurotransmission^13,14, bone homeostasis^15,16, and directly counteract the inhibitory effect of hypothermia on cap-dependent expression thereby promoting global protein synthesis (GPS)¹⁷.

The brain is highly vulnerable to changes in the external temperature during infancy^18,19. Both the fetus in utero and neonates early postpartum, depend on a caregiver for temperature maintenance. Life’s unpredictability precludes a guarantee that a mother can sustain thermoneutrality of the fetus or newborn unabated^20,21. Evidence suggests that CSPs are temporarily increased during early neurodevelopment as molecular safeguards to protect the neonatal brain from changes in external temperature until endogenous mechanisms of thermogenesis mature^22,23. Consequently, baseline brain levels of the major CSPs, RNA-binding motif 3 (RBM3) and cold-inducible RNA-binding protein (CIRBP), are inversely associated with postnatal age in mice, rats, and in humans^22,24–26. Similarly, brain levels of small reticulon-3 (RTN3) isoforms, some of which may also be novel CSPs, are inversely associated with postnatal age in mice and in humans^5,25,27. Moreover, brain morphology is normal in newborn RBM3 KO mice if maintained at normothermia, but pups subjected to hypothermia in utero by exposing the dam to an external cold stress, are born with severe CNS abnormalities²³.

Hypoxia-ischemia (HI) from a sentinel event produces devastating damage to the newborn brain resulting in neonatal encephalopathy (NE)²⁸. Therapeutic hypothermia (TH) to 33°C for 72h, initiated within 6h of birth, is the standard of care to improve long-term neurological outcomes in term newborns with moderate to severe NE^29–31. It is unknown if TH increases neuroprotective CSPs in this and related settings^31,32. Furthermore, it is unknown if HI disturbs the normal neurodevelopmental time course of transient CSP expression in post-resuscitated infants. Here we address this latter question. We first characterized neuropathology in a version of the Rice-Vannucci (RV) model modified to include rigorous temperature monitoring/control in PND10 pups to model perinatal asphyxia in the normothermic term infant, and comprehensively assessed CSPs (RBM3, CIRBP, RTN3) in the normothermic cortex, thalamus, and hippocampus at 1d, 4d, and 8d post-injury, to test the hypothesis that HI alters the neurodevelopmental time course of CSP expression. We provide a foundational dataset using rigorous temperature control to inform the design of future studies on methods to induce and/or modulate neuroprotective CSPs in HI induced NE.

Materials & Methods

Additional details are provided in the Supplementary Methods.

Animals.

Studies were approved by the IACUC of the University of Pittsburgh. Tissues were analyzed at the University of South Florida via a Material Transfer Agreement. C57BL/6 foster dams with litters consisting of 7–8 male pups were purchased from Charles River. Mice on a 12h light/dark cycle had ad libitum access to standard chow.

HI Injury Model.

PND 10 pups were isoflurane anesthetized (3% induction and 1.5–3% maintenance) in a 2:1 mixture of N₂O/O₂ and weighed. Core temperature during sham or occlusion surgery was monitored via a neonatal rectal probe (Physitemp). Body temperature (T_b) was maintained to ~36°C. Post-surgical temperature was measured with a Braun no-touch infra-red pediatric thermometer (Braun Healthcare US). Pilots in naïve PND10 pups (n=6) showed high concordance between temperatures measured by a rectal thermometer (T_b of 36.5±0.73°C) vs. the Braun IR thermometer (Braun IRT) (T_b of 36.6±0.30°C). Normothermia was ~36°C based on pilots in cage-nesting naïve PND10 pups in which T_b was found to be 36.1±0.34°C (n=6). The right common carotid artery (CCA) was accessed by a 0.2–0.3 cm neck incision, sutured and cut. The incision was closed with 3M Vetbond Tissue Adhesive (Maplewood) and Bupivacaine (0.25%) applied to the wound. Mice advanced in blocks of four. A second technician maintained pups at ~36°C in the post-surgical phase by applying heat as needed and by regular T_b monitoring with the Braun IRT. The four-pup block was then returned to the foster dam for 1h. Next, pups advanced to the hypoxia phase and were placed inside a specialized acrylic hypoxia chamber glove box (Coy; Grass Lake, MI). In pilots we observed a high rate of mortality when hypoxia (8% O₂ / 92% N₂) was maintained for >30 mins. Thus, we titrated hypoxia to a duration that produced reproducible brain damage but no mortality, resulting in the 25 min insult level used in this study. An ambient hypoxia chamber temperature setting of 38–40°C maintained neonatal T_b to ~36°C. Shams receive identical procedures without CCA ligation and were placed inside the chamber for 25 min at normoxia (21% O₂ /79% N₂). After hypoxia or normoxia, T_b was managed to ~36°C in room air for 2h:5mins on a benchtop with lamps and heating blankets, and temperature recorded every 15 mins. Pups were then returned to dams until weaning or euthanasia. Cages were supplemented with multiple types of bedding, including Enviro-dri®, to promote high-quality nest construction and decrease thermal stress caused by standard housing^33,34. Details of our approach are provided (Supplementary Fig. 1).

Histology Staining & Neuropathology Assessment.

Anesthetized pups were transcardially perfused with heparinized cold saline, and 4% paraformaldehyde in 4% Sucrose/0.1M Phosphate Buffer/pH 7.2 (Electron Microscopy Sciences). Isolated brains were post-fixed 24h and shipped to Neuroscience Associates (NSA, Knoxville, TN). Brains were embedded in a gelatin matrix (MultiBrain® Technology, NSA). The block was frozen by immersion in chilled 2-Methylbutane and mounted on a freezing stage of an AO 860 sliding microtome. The MultiBrain® block was sectioned coronally through the entire specimen length and collected into a series of 24 cups containing antigen preservation solution (50% PBS pH7.0, 50% Ethylene Glycol, 1% Polyvinyl Pyrrolidone). Free floating sections of an entire cup–each cup having section representation across the full brain rostral to caudal (i.e., a total of 29 “levels”)-were stained with either Fluro-Jade (FJ), GFAP, Iba-1, Weil, or Amino Cupric Silver /Neutral Red Counter. Stained sections were mounted on glass slides. Based on preliminary observations, whole brains at levels 15 and 18, across all brains and stains, were digitally scanned by NSA at 20X magnification on a TissueScope LE120 (Huron Digital Pathology). Data were uploaded to Concentriq (Proscia; Philadelphia, PA) for manual acquisition of regions of interest at 2X, 4X, 10X, and 20X magnification for figure compilation. Slides of Amino Cupric Silver/Neutral Red Counter, Iba1, and GFAP were analyzed by an expert veterinary neuropathologist (RHG) to construct a comprehensive neuroanatomical assessment table of microscopic pathology that spanned all brain levels (1–29). The diagnostic category of “neuropil argyrophilia” was used when a pattern of finely-grained silver deposition was present in the absence (or in addition to) distinct neuronal cell body staining by the amino cupric silver stain³⁵. Neuron argyrophilia scores are based on estimates of the percentage of degenerating (silver positive) vs. normal (unstained) neurons counted in each neuroanatomical region and take into account the size of the brain region (lesions may be focal, multifocal or diffuse). Normal (0) = no stained neurons observed. Minimal (−) = very few neurons judged to be degenerative. Mild (+) = < ~25% of neurons affected. Moderate (++) = ~25–50% of neurons affected. Marked (+++) = ~50–75% of neurons affected. Severe (###) = >75% of neurons affected. All regions characterized by neuron argyrophilia also exhibited reactive microglia and astrocytes characterized microscopically by increased staining of microglial cells (Iba1) or of astrocytes (GFAP). In both processes-designated respectively as microgliosis and astrocytosis-there was also altered morphology of the reactive microglia and astrocytes characterized by thickened more prominent cytologic processes. Foci with severe loss of cellularity (shown with all stains) were classified as “necrosis”. Examples of staining at a single brain level are shown (supplementary figures).

Western Blot.

We used our standard protocol²⁵. (See Supplementary Methods).

Lesion Volume Analysis.

Whole fixed brains were embedded in paraffin. Sections were taken every 0.5mm from posterior to anterior and stained with H&E. Hemispheric area was measured on each slide and summed for total volume (MCID software, St. Catherines, Ontario). Hippocampal area was measured on every slide with a visible hippocampus and the volume was calculated.

Statistical Analysis.

Western blot densitometry of total protein stain (TPS treated membranes) and CSPs, MBP, and cell death targets were measured with UN-SCAN-IT software (Silk Scientific). Each target of interest (densitometry value) was standardized by dividing by the densitometry value of TPS corresponding to the target’s lane. Standardized densitometic values within each blot were then normalized. For CSP/MBP analysis (48/brain region) samples were split evenly across two 26-well criterion gels to ensure equal injury representation (Sham vs. HI) and postnatal age on each blot. Data were analyzed using a 2-Way-ANOVA and Tukey’s multiple comparison post-hoc test. Molecular analysis of cell death pathways at 3d post-injury in hippocampal homogenates were analyzed by an unpaired two-tailed t-test. Data were significant at p<0.05.

Results

Male PND10 pups were subjected to HI or sham manipulations (Supplementary Fig. 1). Neuronal death was assessed by FJ staining at 3, 5, and 7d post-injury. Increased FJ staining was detected in the ipsilateral hippocampus at 3d post-injury (Fig. 1A). FJ staining was generally absent in HI-injured brains 5–7d later (Fig. 1A). Next, cell signaling changes were investigated. The levels of the p75 neurotrophin receptor (P75NTR) and cyclophilin C (CYPC) decreased 3d post-injury in the hippocampus (Fig. 1B, 2D, Supplementary Figs. 2A, 2C). The levels of the pro-apoptotic protein Bax increased (Fig. 1C, Supplementary Fig. 2B). There was no change in the levels of cyclophilin A (CYPA) (Fig. 1E, Supplementary Fig. 2D). Finally, 14d post-injury lesion volume analysis confirmed robust tissue loss in both the ipsilateral hemisphere (Fig. 1F) and ipsilateral hippocampus (Fig. 1G) of injured pups, vs. either the contralateral hemisphere/hippocampus of injured pups or vs. sham ipsilateral hemisphere/hippocampus.

Fig. 1: Neuronal Death in Normothermic HI-Injured Mice.

(A) Fluro jade (FJ) staining in the ipsilateral hippocampus 3d post-injury (n=2 representative mice per time point). (B-E) Densitometric analysis (n=4/group) showed p57NTR and CYPC levels decreased 3d post-injury in the ipsilateral hippocampus. No change in CYPA was detected. Bax was increased 3d post-injury. (F & G). Chronic brain tissue loss was seen in the cortical hemisphere and hippocampus ipsilateral to the CCA ligation (n=11/group sham and n=12/group HI). Data were significant at p <0.05. Box plots show minimum, maximum, interquartile range (IQR), and median. Asterisks in graphs indicate post hoc significance. P75 neurotrophic receptor (p75NTA), cyclophilin C (CYPC), cyclophilin A (CYPA), common carotid artery (CCA).

Fig. 2: Microgliosis & Astrocytosis in Normothermic HI-Injured Mice.

(A-C) Iba-1 staining (purple) and GFAP staining (brown) in the contralateral/ipsilateral hippocampus in a representative mouse at 3d, 5d, and 7d, post-injury. (D-G) 20 X magnification of Iba-1 and GFAP staining in the contralateral vs. ipsilateral hippocampus at 3d post-injury in a representative mouse. Ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP).

A comprehensive neuropathological assessment of microgliosis, astrocytosis, and neuronal degeneration was also performed. Microgliosis and astrocytosis was most severe in the injured hippocampus and detected 3–7d post-injury (Fig 2., Supplementary Figs. 3–4, Table 1). All hippocampal sectors (CA1, CA2, CA3, CA4) had activated microglia and astrocytes. In contrast, in the dentate gyrus (DG), microgliosis and astrocytosis were robustly increased 3d post-injury, but began to decrease thereafter and were minimal by 7d (Fig 2A–G, Table 1). Additional brain regions in some pups showed mild persistent microgliosis and astrocytosis up to 7d post-injury including in temporal, occipital and parietal cortex, and thalamus (Table 1). Neuronal degeneration was generally accompanied by microgliosis and astrocytosis. Neuron argyrophilia was most severe, persisted the longest, and seen in all HI-injured pups in hippocampus (Fig 3., Supplementary Fig. 5, Table 1). Curiously, the magnitude and duration of ongoing neuronal degeneration was greatest in the CA2, followed by the CA3 and CA4. In contrast, neuronal degeneration in the CA1 began to resolve by 7d post-injury.

Table 1:

Neuropathology of the Ipsilateral Hemisphere at 3d, 5d, and 7d Post-HI

	Sham (n=9)	3d HIE (n=3)	5d HIE (n=3)	7d HIE (n=3)		Sham (n=9)	3d HIE (n=3)	5d HIE (n=3)	7d HIE (n=3)
Olfactory Bulb					Thalamus
Neuron Argyrophilia	0	1 (+)	0	0	Neuron Argyrophilia	0	1 (++)	1 (+)	1 (++)
Neuropil Argyrophilia	0	1 (+++)	0	0	Neuropil Argyrophilia	0	1 (++)	0	0
Microgliosis	0	1 (++)	0	0	Microgliosis	0	1 (+++)	1 (+)	1 (++)
Piriform Cortex					Astrocytosis	0	1 (++)	1 (+)	1 (++)
Neuron Argyrophilia	0	l (++)	1 (4)	0	Temporal Cortex
Neuropil Argyrophilia	0	2 (++, +++)	1 (+)	0	Neuron Argyrophilia	0	2 (++,++)	1 (++)	1 (++)
Microgliosis	0	1 (+++)	1 (+)	0	Neuropil Argyrophilia	0	2 (++, +++)	1 (+)	1 (+++)
Astrocytosis	0	1 (++)	1 (+)	0	Microgliosis	0	2 (+++, +++)	1 (+++)	1 (+++)
Necrosis	0	1 (+)	0	0	Astrocytosis	0	2 (+++, +++)	1 (++)	1 (+++}
Frontal Cortex					Necrosis	0	2 (++, ++)	0	0
Neuron Argyrophilia	0	2 (++, +++)	1 (4)	0	Occipital Cortex
Neuropil Argyrophilia	0	2 (++, +++)	1 (+)	0	Neuron Argyrophilia	0	1 (+)	1 (+++)	1 (+++)
Microgliosis	0	2 (+++, +++)	1 (+)	0	Neuropil Argyrophilia	0	2 (+,++)	1 (+++)	1 (++)
Astrocytosis	0	2 (−, +++)	1 (+)	0	Microgliosis	0	0	1 (+++)	1 (++)
Necrosis	0	2 (++, +++)	0	0	Astrocytosis	0	0	1 (+++)	1 (++)
Hippocampus CA1					Necrosis	0	2 (+++, ###)	0	1 (+)
Neuron Argyrophilia	0	1 (−)	2 (+, ++)	1 (+)	Septal Nuclei
Neuropil Argyrophilia	0	0	2 (+,+++)	1 (+)	Neuron Argyrophilia	0	2 (+ ,++)	0	0
Microgliosis	0	2 (++, +++)	2 (+, +++)	1 (+)	Neuropil Argyrophilia	0	1 (+)	0	0
Astrocytosis	0	2 (++, +++)	2 (+, ++)	2 (+, ++)	Microgliosis	0	1 (+)	1 (+)	0
Necrosis	0	2 (+++, +++)	0	0	Astrocytosis	0	1 (++)	1 (+)	0
Hippocampus CA2					Parietal Cortex
Neuron Argyrophilia	0	1 (++)	3 (+, +++, +++)	3 (+, +, +++)	Neuron Argyrophilia	0	1 (+)	1 (+)	1 (+++)
Neuropil Argyrophilia	0	0	3 (+, ++, +++)	3 (+, +, +)	Neuropil Argyrophilia	0	1 (+)	1 (+)	1 (+)
Microgliosis	0	2 (++, +++)	3 (+++, +++, +++)	2 (+++, +++)	Microgliosis	0	1 (++)	1 (+)	1 (+++)
Astrocytosis	0	2 (++, +++)	3 (++, ++, +++)	3 (++, ++, ++)	Astrocytosis	0	2 (++, +++)	1 (+)	1 (+++)
Necrosis	0	2 (+++, +++)	0	0	Necrosis	0	2 (+++, +++)	0	0
Hippocampus CA3					Cortical Atrophy	0	0	0	1 (++)
Neuron Argyrophilia	0	1 (++)	2 (++, +++)	2 (+, +)	Caudate Nucleus/Putamen
Neuropil Argyrophilia	0	0	2 (+, +)	2 (+, +)	Neuron Argyrophilia	0	2 (+, +)	1 (+)	0
Microgliosis	0	2 (++, +++)	2 (+, +++)	1 (+++)	Neuropil Argyrophilia	0	2 (++, +++)	1 (+)	0
Astrocytosis	0	2 (++, +++)	2 (++, ++)	2 (+, ++)	Microgliosis	0	2 (+++)	1 (+)	0
Necrosis	0	2 (+++, +++)	0	0	Astrocytosis	0	2 (+++)	1 (+)	0
Hippocampus CA4					Globus Pallidus
Neuron Argyrophilia	0	0	2 (−, −)	2 (−, +)	Neuron Argyrophilia	0	2 (+, +)	0	0
Microgliosis	0	2 (++, +++)	2 (+, +)	2 (++, +++)	Neuropil Argyrophilia	0	2 (+, ++)	0	0
Astrocytosis	0	2 (+, +++)	2 (++, ++)	2 (++, +++)	Necrosis	0	1 (+)	0	0
Necrosis	0	2 (+++, +++)	0	0	Fimbria
Dentate Gyrus					Microgliosis	0	1 (++)	0	0
Neuron Argyrophilia	0	2 (+, ++)	1 (−)	0	Astrocytosis	0	1 (++)	0	0
Neuropil Argyrophilia	0	0	1 (−)	0	Corpus Callosum
Microgliosis	0	2 (++, +++)	1 (+)	0	Microgliosis	0	1 (++)	0	0
Astrocytosis	0	2 (+++, +++)	1 (++)	1 (−)	Mamillary Bodies
Necrosis	0	2 (+++ +++)	0	0	Neuron Argyrophilia	0	1 (++)	0	0
Entorhinal Cortex					Neuropil Argyrophilia	0	1 (++)	0	0
Neuron Argyrophilia	0	1 (+++)	0	0	Midbrain
Neuropil Argyrophilia	0	1 (++)	0	0	Astrocytosis	0	1 (++)	0	0
Microgliosis	0	1 (++)	0	0	Medial Geniculate
Astrocytosis	0	1 (++)	0	0	Neuron Argyrophilia	0	2 (++, +++)	0	0
Lateral Geniculate					Neuropil Argyrophilia	0	2 (+++, +++)	0	0
Neuron Argyrophilia	0	1 (+)	0	0	Microgliosis	0		0	0
Amygdala					Astrocytosis	0	1 (++)	0	0
Neuron Argyrophilia	0	2 (+ , ++)	0	0	Substantia Nigra Compact
Neuropil Argyrophilia	0	1 (+++)	0	0	Microgliosis	0	2 (++, +++)	0	0
Microgliosis	0	2 (++, +++)	0	0	Substantia Nigra Reticular
Astrocytosis	0	2 (++, +++)	0	0	Neuron Argyrophilia	0	1 (++)	0	0
Retrosplenial Cortex					Neuropil Argyrophilia	0	1 (+)	0	0
Neuron Argyrophilia	0	2 (+, +++)	0	0	Microgliosis	0	1 (+++)	0	0
Neuropil Argyrophilia	0	2 (++, +++)	0	0	Astrocytosis	0	1 (++)	0	0
Microgliosis	0	1 (+++)	0	0	Superior Colliculus
Astrocytosis	0	1 (++)	0	0	Neuron Argyrophilia	0	1 (+++)	0	0
Necrosis	0	1 (+)	0	0	Neuropil Argyrophilia	0	1 (+++)	0	0
Subiculum					Inferior Colliculus
Neuron Argyrophilia	0	2 (+, +++)	0	0	Neuron Argyrophilia	0	1 (++)	0	0
Neuropil Argyrophilia	0	2 (+++, +++)	0	0	Microgliosis	0	1 (+)	0	0
					Astrocytosis	0	1 (++)	0	0

^*Diagnosis Based on Amino-Cupric Silver, Iba-1, and GFAP Staining

^**Neuropathology Scores: Histologically Normal (0), Minimal (−), Mild (+), Moderate (++), Marked (+++), and Severe (###) Evidence of Injury

^***The numerical value in each box indicates the number of brains showing evidence of injury in each region/category and their associated score in ().

Fig. 3: Neuronal Degeneration in Normothermic HI-Injured Mice.

(A, C, and E) Amino Cupric Silver Staining (black) and Congo red counterstaining (pink) in brain sections from two representative mice at 3d, 5d, and 7d, post-injury. (B) 4 X magnification of silver staining in the hippocampus and corpus callosum 3d post-injury. (D) 10 X magnification of CA1 hippocampal neuron degeneration 5d post-injury. (G) 4 X magnification of silver staining in the thalamus and cortices 3d post-injury. (F) 10 X magnification of CA3 hippocampal neuron degeneration at 7d post-injury.

Next, we assessed thalamic white matter injury. Myelin-basic protein (MBP) levels were measured in protein extracts from the isolated thalamus at 24h, 4d, and 8d post-injury. MBP levels were below detection limits in PND11 sham and in injured pups (Supplementary Fig 6A, 6B). In contrast, MBP levels were robustly detected in the thalamus by PND18 (Supplementary Fig 6). Injured pups showed a nonsignificant (p=0.06) trend for decreased thalamic MBP levels. Weil’s myelin stain further supported myelin sheath damage in the thalamus in some HI injured pups. Myelin staining (deep blue/black) developmentally increased in the thalamus from PND13-PND17 (Supplementary Figs 6C–E, 7), but two of three HI-injured pups at the 7d post-injury time point showed decreased Weil staining in the ipsilateral thalamus and cerebral peduncles (cpd) (Supplementary Fig 6E–G).

We next tested the hypothesis that HI alters the neurodevelopmental expression of RBM3, CIRBP, and RTN3. All three CSPs robustly decreased in the cortex with advancing age in shams (Fig. 4, Supplementary Figs. 8, 10A). Cortical RBM3 levels were not affected by HI-injury, nor was there an interaction with age (Fig. 4A–B, Supplementary Fig. 8A–B). Cortical CIRBP levels showed a significant interaction with age and injury (Fig. 4C–D and Supplementary Fig. 8C–D). Cortical RTN3 levels were significantly affected by injury and showed a non-significant trend toward a potential interaction with age (Fig. 4E–F, Supplementary Fig. 8E–F). Specifically, a post-hoc significant increase in RTN3c levels was detected 24h post-injury (Fig. 4F).

Fig. 4: Effect of HI on CSP Levels in the Cortex.

(A, C, & E) Representative western blot (n=4/group) of cortical RBM3, CIRBP, and RTN3 levels in shams vs. HI-injured pups 24h, 4d, and 8d post-injury. (B, D, & F) Densitometric analysis of cortical RBM3, CIRBP, and RTN3 levels (n=8/group). Total protein stains are available in the supplementary. Data were significant at p <0.05. Box plots show minimum, maximum, interquartile range (IQR), and median. Asterisks in graphs indicate post hoc significance. RNA-binding motif 3 (RBM3), cold inducible RNA-binding protein (CIRBP), reticulon 3 (RTN3).

In the thalamus all three CSPs decreased with advancing age in shams but were unaffected by HI injury (Fig. 5, Supplementary Figs. 9, 10B). Thalamic RBM3 levels were not affected by HI-injury, nor was there an interaction with age (Fig. 5A–B, Supplementary Fig. 9A–B). Thalamic CIRBP levels were not affected by HI-injury, nor was there an interaction with age (Fig. 5C–D, Supplementary Fig. 9C–D). Thalamic RTN3 levels were not affected by HI-injury, nor was there an interaction with age (Fig. 5E–F, Supplementary Figs. 9E–F, 10B).

Fig. 5: Effect of HI on CSP Levels in the Thalamus.

(A, C, & E) Representative western blot (n=4/group) of thalamic RBM3, CIRBP, and RTN3 levels in shams vs. HI-injured pups 24h, 4d, and 8d post-injury. (B, D, & F) Densitometric analysis of thalamic RBM3, CIRBP, and RTN3 levels (n=8/group). Total protein stains are available in the supplementary. Data were significant at p <0.05. Box plots show minimum, maximum, interquartile range (IQR), and median. Asterisks in graphs indicate post hoc significance. RNA-binding motif 3 (RBM3), cold inducible RNA-binding protein (CIRBP), reticulon 3 (RTN3).

In the hippocampus there were marked differences in CSP detection (Fig. 6, Supplementary Figs. 11–12). Hippocampal RBM3 levels were readily detectable, decreased in shams with advancing age, and showed a significant interaction with injury (Fig. 6A–B, Supplementary Fig. 11A–B). RBM3 levels increased 8d post-injury in HI-injured pups vs. shams but did not reach significance on post-hoc analysis. (Fig 6B). CIRBP was readily detectable, decreased in shams with advancing age, and had a significant interaction with injury manifested by a slight increase at 8d post-injury, but which was not significant on post-hoc analysis. (Fig. 6C–D, Supplementary Fig. 11C–D). Hippocampal RTN3 and RTN3c were faintly detected (Supplementary Fig. 12). The absence of RTN3 in most samples precluded meaningful densitometric analysis. RBM3 has been shown to regulate RTN3 expression, thus, we performed a spearman correlation on CIRBP or RTN3 vs. RBM3 levels, across all brain tissues/ages. In the cortex, RBM3/CIRBP was highly correlated in shams (Fig. 6E). and maintained after injury (Fig. 6F). RBM3/RTN3 were also correlated in shams, but not after HI injury (Fig. 6E, 6F). The same phenomenon was observed in the thalamus (Fig. 6G, 6H). Lack of RTN3 expression in the hippocampus precluded a correlation analysis, but hippocampal RBM3/CIRBP levels were correlated in shams and after injury (Fig. 6I, 6J).

Fig. 6: Effect of HI on CSP Levels in the Hippocampus.

(A & C) Representative Western blot (n=4/group) of hippocampal RBM3 and CIRBP levels in shams vs. HI-injured pups 24h, 4d, and 8d post-injury. (B & D) Densitometric analysis of hippocampal RBM3 and CIRBP levels (n=8/group). Total protein stains are available in the supplementary. (E-J) Spearman correlation analysis of RBM3 vs. CIRBP and RTN3 levels in shams and HI-injury pups across all time points in cortex, thalamus, and hippocampus. Data were significant at p <0.05. Box plots show minimum, maximum, interquartile range (IQR), and median. Asterisks in graphs indicate post hoc significance. RNA-binding motif 3 (RBM3), cold inducible RNA-binding protein (CIRBP), reticulon 3 (RTN3).

Finally, we investigated if inter-subject differences in insult severity correlated with changes in RBM3 and CIRBP levels in the injured hippocampus. Here, GFAP and Iba1 protein levels were used as surrogate markers of insult severity (Supplementary Figs. 13–15). GFAP and Iba1 levels were significantly increased at 4d and 8d post-HI but showed high inter-subject variability (Supplementary Figs. 13C–14C). Hippocampal RBM3 levels did not correlate with GFAP levels (Supplementary Fig. 13D–E) or Iba1 levels (Supplementary Fig. 14D–E) on 4d or 8d post-injury. In contrast, hippocampal CIRBP levels at 4d post-injury, but not at 8d, showed a significant negative correlation with GFAP levels (Supplementary Fig. 13F–G) and Iba1 levels (Supplementary Fig. 14F–G).

Discussion

The RV model of unilateral ischemia is the gold-standard to study preclinical HI-mediated neuropathology in neonatal rodents. Based on neurodevelopmental metrics, a PND 7–10 pup approximates a 36–40-week term newborn³⁶. Here we established a modified RV model in PND 10 mice which incorporates non-invasive temperature surveillance to rigorously maintain target temperature across the surgical, hypoxic, and post-resuscitation phases. This ensured that brief acute endogenous neuroprotective hypothermia did not manifest immediately after injury as reported³⁷.

Neuronal Death & Brain Injury Markers in Temperature Managed PND 10 HI-Injured Mice.

The RV model results in neuronal apoptosis and necrosis³⁸. FJ staining increased in the HI-injured hippocampus 3d post-injury but was sporadic-to-absent 5–7d later. FJ signal positivity is linked to eosinophilic necrosis or “red neurons”³⁹. The limited FJ staining here mimics reports that fewer neurons show evidence of eosinophilic-positive necrotic death in the neonatal RV model (although other types of necrosis are readily detected), vs. adult models of brain HI³⁸. However, the detection of FJ signals at 3d post-injury in our model, suggests that it has utility in assessing the efficacy of neuroprotective therapies at that time point

Next, we screened for potential HI-injury markers at 3d post-injury in hippocampal homogenates. P75NTR robustly decreased after HI. Studies show that neuronal KO of p75NTR disrupted neurodevelopment in mice and led to a profound decrease in cortical and hippocampal volumes by ~40% and ~35%, respectively, vs. WTs⁴⁰. The pro-apoptotic protein Bax modestly increased 3d post-injury, consistent with reports by others showing that Bax inhibiting peptides protect in the RV model⁴¹. Finally, we observed a robust decrease in CYPC. This was specific as we did not observe a similar change in CYPA post-HI.

Lastly, we measured 14d hemispheric and hippocampal lesion volume⁴². Significant brain tissue volume loss was detected ipsilateral to CCA-ligation. Together the findings confirm robust neuronal death in the acute and sub-acute phases after HI in our modified RV model.

Gliosis in Temperature Managed PND 10 HI-Injured Mice.

Astrocytosis and microgliosis are secondary injury mechanisms in perinatal asphyxia, and molecular signals released by these processes have clinical prognostic value for assessing HI severity^43,44. GFAP and Iba-1 robustly increased in ipsilateral hippocampus 3–7d post-injury. Temporal changes in microglial staining intensity agree with other studies in the RV model⁴⁵. The small sample here (n=3/group for injured time points) is a limitation. However, we sought to confirm the expected patterns of neuropathology studied by others, now with highly rigorous temperature control, before studying CSPs.

White Matter Injury in Temperature Managed PND 10 HI-Injured Mice.

White matter injury is common on neuroimaging in infants with moderate/severe HI and is associated with damage to the basal ganglia and thalamus⁴⁶. Studies in the RV model suggest that thalamic neurodegeneration blossoms in the first week^38,47. We characterized white matter damage with Weil stain and MBP immunoblotting. MBP robustly increased in shams by PND 18, and at which time HI-injured pups began to manifest a trend toward decreased levels 8d post-injury (p=0.06). Weil stain of the myelin sheath in ipsilateral thalamus and cerebral peduncles also showed a decrease in staining 7d post-injury.

Tissue Degeneration in Temperature Managed PND10 HI-Injured Mice.

Cupric-silver detects degenerating synaptic terminals, cell bodies, and dendrites at 1–4d post-injury, whereas axonal degeneration is detected 3–7d post-injury⁴⁸. Silver staining robustly increased throughout the ipsilateral hemisphere at 3d post-HI, including in the cortex, hippocampus, and thalamus, but with little evidence of delayed diffuse axonal degeneration at 5d-7d post-injury. Patchy dendritic and neuropil cupric-silver positivity persisted 3–7d across hippocampal subfields, which is indicative of ongoing delayed neuronal death/degeneration. This is consistent with HI in term infants from a perinatal asphyxia and contrasts the diffuse non-cystic white matter injury/axonal-damage characteristic of encephalopathy of prematurity^49–51.

Effect of HI on CSP Expression in the Developing Normothermic Brain.

Homeothermy in newborn mice proceeds in three phases⁵². Pups aged PND 0–7 are functionally poikilothermic (Phase I). Pups aged PND 8–14 have limited thermogenesis (Phase II). Pups aged PND 15–17 reach peak (adult) capacity for shivering thermogenesis and can maintain body temperature (Phase III)⁵². RBM3, CIRBP, and RTN3 are abundant in the mammalian normothermic brain during infancy, then rapidly decrease at an early postnatal age. We examined if HI altered the baseline CSP expression in brain during phases II-III of the thermogenic transition in neonatal mice. CSPs were measured at PND 11 (early Phase II), PND 14 (late Phase II), and PND 18 (late Phase III). Baseline brain CSP levels in shams were inversely associated with the phase of thermoregulatory development. Whether this relationship is causative or correlative is unclear. Given that hypoxia alone can increase RBM3 and CIRBP levels, surprisingly HI in normothermic pups minimally impacted the age-related decline in brain CSPs⁵³.

Cold-induced RBM3 is a potent neuroprotectant in vivo^10,11. RBM3 KO mice subjected to cold-stress in utero have postnatal brain deformities, suggesting that it safeguards the developing CNS from potentially detrimental antenatal hypothermia²³. Furthermore, in HI-injured RBM3 KOs, neural stem cell proliferation in the subgranular zone (SGZ) of the DG was severely impaired and apoptosis was increased vs. WTs⁵⁴. We found that age was associated with a significant decrease in RBM3 levels in the cortex, thalamus, and hippocampus. However, HI did not affect RBM3 levels in the cortex or thalamus. In contrast, an interaction of HI with age was detected in the hippocampus, manifested as increased levels 8d post-injury. Intriguingly, surrogate markers of insult severity (GFAP, Iba1) did not correlate with RBM3 levels in the hippocampus at 4d and 8d post-injury. That raises important questions/concepts for additional exploration. Seventy-two hours of TH within 6h of birth is standard of care to treat HI in term newborns in high-income countries and decreases long-term disability³⁰. However, it is unknown if clinically optimized TH slows the age-related loss of RBM3 expression in the developing brain post-insult⁵⁵. Similarly, the optimal depth and duration of cooling that maximally increases/prolongs brain RBM3 levels in the RV model needs to be explored. The lack of a correlation between GFAP/Iba1 and RBM3 levels supports the notion that RBM3-inducing therapies should be studied across insult severity. Characterization here on neurodevelopmental timing of RBM3 changes early after HI, in the RV model, provides insight for future studies.

CIRBP can promote neuroprotection and neurotoxicity². Whether CIRBP-mediated effects on neuronal survival are temperature-dependent or modified by developmental age remains to be determined. In adult mice, infarct volume is decreased in normothermic CIRBP KOs after middle cerebral artery occlusion⁸. Furthermore, studies in normothermic BV-2 cells suggested that hypoxia induces microglial CIRBP secretion and impacts neuronal injury⁸. We found that CIRBP levels in the cortex, thalamus, and hippocampus were associated with neonatal age. In thalamus and hippocampus, the age-mediated decline was modest. In cortex, the decrease was steep mirroring the rapidly fading expression of RBM3 in cortex and hippocampus by PND 18. Furthermore, an interaction (injury vs. age) was detected in the cortex and hippocampus, manifested as a slight decrease in HI-injured CIRBP levels at 4d post-injury but a slight increase at 8d post-injury. Also, surrogate markers of insult severity (GFAP, Iba1) correlated with hippocampal CIRBP levels at 4d post-injury. Unaddressed questions include-does TH increase CIRBP levels in the RV model, is it neuroprotective/neurotoxic, what depth/duration of TH optimally induces (or avoids) CIRBP induction, and might early/acute serum CIRBP levels serve as a HI severity biomarker? We did not measure serum CIRBP levels to know if the insult-severity-dependent decrease in brain levels was associated with increased serum levels. However, serum CIRBP is increased in patients with brain injury from intracerebral hemorrhage; it may also be detectable post-injury in the clinical NE population and merits investigation⁵⁶.

RTN3 is a recent addition to the small family of CSPs. Like CIRBP, it may be neuroprotective and/or neurotoxic². However, chronically increasing levels at normothermia can produce RTN3-immunoreactive dystrophic neurites¹⁴. RBM3 also regulates RTN3 induction during cold-stress⁵. We found that age was associated with decreased RTN3 levels (25 kDa and 20 kDa forms) in cortex and thalamus. RTN3 in hippocampus was at the limit of detection, precluding analysis; however, hippocampal RTN3 was reported to increase with age from 2–18mo in mice⁵⁷. In cortex, injury independently associated with an increase in RTN3c levels. Its impact on post-HI recovery remains unclear. Finally, we measured the correlation between RBM3/CIRBP (classic CSPs) and RBM3/RTN3 expression for additional insights into their possible co-regulation. In all three brain regions, RBM3/CIRBP were strongly correlated in shams and after HI. RBM3/RTN3 showed weaker correlations in shams. However, after HI, RBM3/RTN3 were no longer correlated. The latter finding raises the possibility that HI-injury may modify downstream signaling pathways that link RBM3 with RTN3 induction⁵. It remains to be tested if this could translate to a decreased ability of TH to induce RTN3 in neonatal HI.

Our study has limitations, specifically, the exclusion of females and the small sample size. Sex-differences are well described in the NE literature; males tend to have larger lesion size vs. females in the RV model^58,59. Also, RBM3 is located on the X chromosome and is among a few genes that escapes X-chromosome inactivation in human female cells^60–62. It is unclear if this phenomenon alters the dynamics of RBM3 expression in the normothermic developing female vs. male brain, or if it helps explain observations in terms of why mild TH may offer greater neuroprotection in juvenile female vs male rodents⁶³. Given the need to improve outcomes in males and females, future studies of the impact of TH on CSPs are warranted in females, and to assess the ability of modulation of these CSPs to provide neuroprotection across temperature and age⁶⁴.

In summary, we investigated the effect of HI on the neurodevelopmental expression of CSPs at normothermia during a critical time window involving the rapid maturation of thermogenesis in neonatal mice. CSPs rapidly decreased in the neonatal brain as heat-generating mechanisms become operational and the levels were not importantly altered by HI. Given the previously reported neuroprotective effects of RBM3 in adult mice¹¹, and ongoing efforts to optimize care in perinatal asphyxia⁶⁵, our findings support the need to determine if blunting the age-related decline in neuroprotective RBM3 (such as with TH) may promote neuroprotection during critical periods post-NE.

Impact.

• The Rapid Decrease in Endogenous Neuroprotective Cold-Shock Proteins (CSPs) in the Normothermic Cortex, Thalamus, and Hippocampus from Postnatal Day (PND) 11–18, Coincides with the Timing of Thermogenesis Maturation in Neonatal Mice.

• Hypoxia-Ischemia (HI) has a Minor Impact on the Normal Age-Dependent Decline in Brain CSP Levels in Neonates Maintained Normothermic Post-Injury.

• HI Robustly Disrupts the Expected Correlation in RNA-Binding Motif 3 (RBM3) and Reticulon-3 (RTN3).

• The Potent Neuroprotectant RBM3 is not Increased 1–4d After HI in a Mouse Model of Neonatal Encephalopathy (NE) in the Term Newborn and in which Rigorous Temperature Control Prevents the Manifestation of Endogenous Post-Insult Hypothermia.