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. 2021 Dec 7:9:737319.
doi: 10.3389/fcell.2021.737319. eCollection 2021.

Early Thalamic Injury After Resuscitation From Severe Asphyxial Cardiac Arrest in Developing Rats

Hoai T Ton  1 , Katherine Raffensperger  1 , Michael Shoykhet  1
Affiliations

Early Thalamic Injury After Resuscitation From Severe Asphyxial Cardiac Arrest in Developing Rats

Hoai T Ton et al. Front Cell Dev Biol. .

Abstract

Children who survive cardiac arrest often develop debilitating sensorimotor and cognitive deficits. In animal models of cardiac arrest, delayed neuronal death in the hippocampal CA1 region has served as a fruitful paradigm for investigating mechanisms of injury and neuroprotection. Cardiac arrest in humans, however, is more prolonged than in most experimental models. Consequently, neurologic deficits in cardiac arrest survivors arise from injury not solely to CA1 but to multiple vulnerable brain structures. Here, we develop a rat model of prolonged pediatric asphyxial cardiac arrest and resuscitation, which better approximates arrest characteristics and injury severity in children. Using this model, we characterize features of microglial activation and neuronal degeneration in the thalamus 24 h after resuscitation from 11 and 12 min long cardiac arrest. In addition, we test the effect of mild hypothermia to 34°C for 8 h after 12.5 min of arrest. Microglial activation and neuronal degeneration are most prominent in the thalamic Reticular Nucleus (nRT). The severity of injury increases with increasing arrest duration, leading to frank loss of nRT neurons at longer arrest times. Hypothermia does not prevent nRT injury. Interestingly, injury occurs selectively in intermediate and posterior nRT segments while sparing the anterior segment. Since all nRT segments consist exclusively of GABA-ergic neurons, we asked if GABA-ergic neurons in general are more susceptible to hypoxic-ischemic injury. Surprisingly, cortical GABA-ergic neurons, like their counterparts in the anterior nRT segment, do not degenerate in this model. Hence, we propose that GABA-ergic identity alone is not sufficient to explain selective vulnerability of intermediate and posterior nRT neurons to hypoxic-ischemic injury after cardiac arrest and resuscitation. Our current findings align the animal model of pediatric cardiac arrest with human data and suggest novel mechanisms of selective vulnerability to hypoxic-ischemic injury among thalamic GABA-ergic neurons.

Keywords: GABA-ergic interneuron; cardiac arrest; hypoxia; ischemia; microglia; neuronal degeneration; reperfusion; thalamic reticular nucleus.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Procedure for asphyxia cardiac arrest and resuscitation experiment following by tissue processing for histological study (A) Timeline and procedure for anesthetic washout, asphyxia, PCR and post-ROSC periods. (B) Experimental procedure for histological staining and analyzing after 24 h after insults. ABP: artery blood pressure; ECG: electrocardiogram; CA: cardiac arrest; CPR: cardiopulmonary resuscitation; ROSC: return of spontaneous circulation; MAP: mean arterial pressure.
FIGURE 2
FIGURE 2
Coronal sections of CuAg and Iba1 labeling from sham and CA rats. The whole brain immunohistochemistry labelling Iba-1 and Amino Cupric Silver (CuAg) show the profound neuronal degeneration and microglial activation, respectively, in the specific region within the thalamus from 12.5 min CA compared to sham rats. The black arrows indicate the location of CA-induced injury in the thalamus, nRT.
FIGURE 3
FIGURE 3
CA induces pronounced microglial activation and neurodegeneration in the thalamic reticular nucleus (A,B) Representative photomicrographs of Iba1 and Amino Cupric Silver (CuAg) stains in the reticular nucleus 24 h after 11; 12; 12.5 min CA and sham-operated rats (C) Schematic illustration of the reticular nucleus, the blue rectangle indicates the captured in A and B; the blue crescent-shape indicates the analyzed region. (D–F) The bar graphs show percentage of Iba1 stained area, CuAg-stained area, and the number of CuAg-stained cell in the ROI. It can be noted that both Iba1 and CuAg stains in RT from the CA rats show a remarkable increase compared to sham (Nested one-way ANOVA with Dunnett’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs sham group; Data are presented as individual values with median and interquartile range. Both left and right RT of 2,3 stained slides from each of 3–6 animals/group were analyzed). Scale bars represent 500 μm (low-power images) and 20 μm (high-power images).
FIGURE 4
FIGURE 4
CA-induced microglia activation and neurodegeneration are observed in the posterior and intermediate but not in the anterior RT (A) Schematic identification of anteroposterior position and shape indicated with respect to bregma. The blue box (2000 μm × 1,600 μm) indicates the captured RT segments; the black arrowheads indicate the location of ROI (B,C) Composition of three Iba1-stained (B) and CuAg-stained (C) coronal sections through the anterior, intermediate and posterior of RT from each of the sham; 11 min, 12 min, and 12.5 min + hypothermia CA groups. Scale bar represents 200 µm applied for all images.
FIGURE 5
FIGURE 5
Gad67-stained and CuAg-stained sections from the same brain regions of the same representative sham or insult rats (A) The intermediate RT from sham section with lacking silver staining (left) shows prominent Gad67+ neurons while RT from CA section (B) with prominent CuAg+ stain in soma show a remarkable reduction of Gad67+ neurons. Scale bars represent 200 µM.
FIGURE 6
FIGURE 6
CA reduces GABAergic interneurons in the RT (A) Coronal Gad67-stained sections at anterior, intermediate and posterior RT from one representative brain in each of sham (upper images) and 12.5 min CA + hypothermia rats (lower images). The blue rectangles (250 × 300 µm) capture the high magnification showed in (B) and analyzed Gad67+ cells in (C). There is statistically significant decrease in the number Gad67-labeled neurons in intermediate RT between CA group compared with sham-operated group (Nested t-test, ∗∗p < 0.01). Data are presented as individual values with median and interquartile range from two to three stained slides from each of three sham and 5 CA animals.
FIGURE 7
FIGURE 7
CA-induced synaptic degeneration in sub-areas of thalamic relays. The ROI scheme (left), CuAg stain in sham (middle) and 12.5 min cardiac arrest (right) in the mediodorsal thalamic nucleus (A), ventroposteriomedial (B), and medial geniculate thalamic nucleus (C). Note that the degenerating synapses were seen in both VPM and MGN but not in MD of injured animals.
FIGURE 8
FIGURE 8
Analysis of GABA-ergic interneurons in the cortex of sham and CA (A) Representative images of Gad67 staining in the sub-regions of cortex from sham (upper images) and CA (lower images). Blue arrows indicate the Gad67-labelled cells counted manually using multi-point function in FIJI (B) Coronal sections at bregma 3.24 mm with regions of interest marked by blue boxes: motor cortex (MC), somatosensory cortex (SS), and auditory cortex (Aud) (C) The graphs show the numbers of Gad67-stained cell with median and interquartile range in the ROIs from three stained slides from three sham and 5 12.5 min CA animals. There is no significant deference in the number Gad67-labeled neurons between CA group compared with sham-operated group (Nested t-test, p > 0.05).
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