Neurodegeneration, Myelin Loss and Glial Response in the Three-Vessel Global Ischemia Model in Rat

(1) Background: Although myelin disruption is an integral part of ischemic brain injury, it is rarely the subject of research, particularly in animal models. This study assessed for the first time, myelin and oligodendrocyte loss in a three-vessel model of global cerebral ischemia (GCI), which causes hippocampal damage. In addition, we investigated the relationships between demyelination and changes in microglia and astrocytes, as well as oligodendrogenesis in the hippocampus; (2) Methods: Adult male Wistar rats (n = 15) underwent complete interruption of cerebral blood flow for 7 min by ligation of the major arteries supplying the brain or sham-operation. At 10 and 30 days after the surgery, brain slices were stained for neurodegeneration with Fluoro-Jade C and immunohistochemically to assess myelin content (MBP+ percentage of total area), oligodendrocyte (CNP+ cells) and neuronal (NeuN+ cells) loss, neuroinflammation (Iba1+ cells), astrogliosis (GFAP+ cells) and oligodendrogenesis (NG2+ cells); (3) Results: 10 days after GCI significant myelin and oligodendrocyte loss was found only in the stratum oriens and stratum pyramidale. By the 30th day, demyelination in these hippocampal layers intensified and affected the substratum radiatum. In addition to myelin damage, activation and an increase in the number of microglia and astrocytes in the corresponding layers, a loss of the CA1 pyramidal neurons, and neurodegeneration in the neocortex and thalamus was observed. At a 10-day time point, we observed rod-shaped microglia in the substratum radiatum. Parallel with ongoing myelin loss on the 30th day after ischemia, we found significant oligodendrogenesis in demyelinated hippocampal layers; (4) Conclusions: Our study showed that GCI-simulating cardiac arrest in humans—causes not only the loss of pyramidal neurons in the CA1 field, but also the myelin loss of adjacent layers of the hippocampus.


Introduction
White-matter injury-along with neuronal loss-is an integral part of the pathologic processes that accompany cerebrovascular diseases. Poor white-matter recovery significantly affects the long-term outcome after an acute stroke [1], impairs sensorimotor function and causes profound neurobehavioral and cognitive impairment [2]. The main function of myelin, which is to increase the speed with which electrical impulses propagate along the myelinated fiber, is well known. Other functions of myelinating

Neuronal Loss and Neurodegeneration in the Hippocampus, Neocortex and Thalamus
The most prominent neuronal loss was observed in the CA1 layer of pyramidal neurons. On Day 10 after GCI, the number of NeuN+ cells in the CA1 field decreased significantly (p < 0.001) compared to the sham-operated animals only in SP (Figure 1c). The layer appears rarefied with the integration of NeuN-negative small-nuclear cells, probably, activated microglia as revealed by previous studies [17,32]. On Day 30 after GCI in the CA1 field SP the number of NeuN+ cells significantly decreased both in comparison with the sham-operated control and the 10-day group (Figure 1b). At this time point only separate pyramidal NeuN+ neurons are observed. Some pyramidal neurons are very weakly stained ( Figure 1a). On average, the number of pyramid neurons in SP 30 days after 7 min of GCI decreased by about 14 times (from 3538 cells per mm 2 in the sham-operated animals to 245 cells per mm 2 in animals from the 30-day group). Other layers of the hippocampus in the CA1 field did not show significant difference compared with controls in the number of NeuN+ cells (Figure 1b).
A combination of Fluoro-Jade C (FJC) and NeuN staining showed that 53.4% remaining pyramidal neurons in the hippocampus on Day 10 after GCI undergo degeneration (Figure 1c,e). On Day 30 the percentage of double-labeled FJC+\NeuN+ cells from surviving neurons was significantly (p < 0.05) reduced compared to the 10-day group, but still remain high (39.2%). In addition, neurodegeneration was observed in the neocortex ( Figure S1) and thalamus ( Figure S2). On Day 10 after GCI in the motor and somatosensory cortex small amount of degenerating neurons was found only in layers II-III. On the 30th day after GCI neurodegeneration was observed not only in layers II-III, but also extended to layer IV. In the thalamus, few degenerating neurons only in the paraventricular thalamic nucleus were found on Day 10 after ischemia. At the 30-Day time point neurodegeneration involved, in addition to the paraventricular thalamic nucleus, neighboring mediodorsal, intermediodorsal thalamic nuclei and habenular nucleus. comparison of NeuN+ cells in the CA1 field between sham-operated controls and animals 10 and 30 days after GCI; (e) percentage of FJC+\NeuN+ cells from NeuN-positive neurons in the CA1 field in sham-operated controls and animals 10 and 30 days after GCI. Significant differences relative to the sham-operated group, according to ANOVA after LSD correction for multiple comparisons: *-p < 0.05, ***-p < 0.001.

Inflammation and Specific Changes of Microglial Morphology After GCI
In animals that underwent GCI, an accumulation of activated microglia was observed in all hippocampal layers at the CA1 field level except the SE (Figure 2a). On the 10th day after GCI in SO, and double-labeled Fluoro-Jade C (FJC)\NeuN-positive (c) neurons in the CA1 field of the hippocampus of sham-operated controls and animals 10 and 30 days after GCI; (d) comparison of NeuN+ cells in the CA1 field between sham-operated controls and animals 10 and 30 days after GCI; (e) percentage of FJC+\NeuN+ cells from NeuN-positive neurons in the CA1 field in sham-operated controls and animals 10 and 30 days after GCI. Significant differences relative to the sham-operated group, according to ANOVA after LSD correction for multiple comparisons: *-p < 0.05, ***-p < 0.001.

Inflammation and Specific Changes of Microglial Morphology after GCI
In animals that underwent GCI, an accumulation of activated microglia was observed in all hippocampal layers at the CA1 field level except the SE (Figure 2a). On the 10th day after GCI in SO, the processes of the cells became thicker in a large part of the cells; the number of the processes increased. In the SP and SL, all Iba1+ cell bodies became larger, the processes were shorter and thicker. In the SP, the accumulation of Iba+ cells is limited to the CA1 field, where they are located everywhere between the pyramidal neurons (Figure 2b,c). In the SR, cells acquired a distinct rod-like morphology: a strongly elongated cell body and an elongated nucleus; numerous short processes depart from the cell body (Figure 2b). Double immunostaining with the myelin basic protein (MBP) and Iba1 showed that the elongated microglial bodies in the SR are oriented predominantly in the same direction as the myelinated fibers in this layer (Figure 2d).  On the 30th day after GCI in all layers of the hippocampus, except the SE, Iba1+ cells showed activated morphology with hypertrophied cell bodies and short thick processes. Quantification of the number of Iba1+ cells in the hippocampal layers showed that on the 10th and 30th day after GCI, the number of cells in all layers (SO, SP, SR, SL) increased significantly (p < 0.001) compared to the control, except the SE (Figure 2i). Thus, after both 10 and 30 days after GCI, only the SE Iba1+ cells did not respond to ischemic damage: their number and morphology did not change compared to the data in the control group of animals (Figure 2b,i).

Astrogliosis
The GCI-induced astrogliosis in the hippocampus is observed at Day 10 and continues to the 30th day in postischemic animals ( Figure 3a). A significant part of GFAP-positive cells in hippocampal layers of postischemic animals showed morphologic changes characteristic of activated astrocytes, such as an enlarged body, thickening of the processes and an increase in their number (Figure 3b). Astrocyte activation after GCI, however, was not observed in all hippocampal layers. Ten days after GCI, no significant changes in GFAP-positive cell morphology were observed in the hippocampus layers of SO, SP, SE, but activated astrocytes with hypertrophied bodies and thickened processes appeared in SR and SL ( Figure 3b). The astrocytes located between the myelin fibers in the SR maintained the radial orientation of their processes (Figure 3c). At the same time point, the number of astrocytes significantly increased when compared to the control group in the layers of SO, SP and SR (p < 0.05-0.001). The number of astrocytes in SL and SE tended to increase, but not statistically significantly (Figure 3d). At Day 30 after GCI, astrocytes cells in all layers of the hippocampus except SE had hypertrophic bodies and short processes, especially in the SR and SL layers ( Figure 3b). Their number significantly increased compared with the sham-operated group in all hippocampal layers (p < 0.05-0.001) (Figure 3d). In sham-operated rats, astrocyte morphology remained unaffected: a small cell body with long thin processes.
layers of SO, SP and SR (p < 0.05-0.001). The number of astrocytes in SL and SE tended to increase, but not statistically significantly (Figure 3d). At Day 30 after GCI, astrocytes cells in all layers of the hippocampus except SE had hypertrophic bodies and short processes, especially in the SR and SL layers ( Figure 3b). Their number significantly increased compared with the sham-operated group in all hippocampal layers (p < 0.05-0.001) (Figure 3d). In sham-operated rats, astrocyte morphology remained unaffected: a small cell body with long thin processes.  (d) quantification of GFAP-positive astrocytes in the hippocampal layers in the sham-operated and postischemic animals, at day 10 and 30 after GCI. Significant differences between the groups, according to ANOVA after LSD correction for multiple comparisons: ***-p < 0.001; **-p < 0.01; *-p < 0.05.

GCI Causes Myelin and Oligodendrocyte Loss in the SO, SP and SR the Hippocampus
GCI caused myelin loss in all hippocampal layers excluding the SL end SE. The most prominent changes in myelination (MBP-positive area) after GCI were found in the SO and SP (Figure 4a,c). In the sections obtained both at Days 10 and 30 after GCI observed a decrease in the number of myelinated fibers, especially at the top part of the SP. The SR was also affected by GCI: in postischemic animals, myelinated fibers in this layer became fragmented. In sham-operated animals, most of the myelinated fibers in the SR were parallel to each other and perpendicular to the SP. Separate fibers can be traced throughout the entire SR, from the SP to SL, unlike the myelin fibers obtained from postischemic animals. A quantitative comparison of animal groups confirms that the SO and SP are most affected by GCI among hippocampal layers. In the SO and SP, the significant differences in the MBP-positive area were found both between the control and the 10-day time point and between the control and the 30-day time point. In addition, a significant (p < 0.05) decrease in the MBP-positive area on the 30th day after ischemia was found in the SR layer compared to the control (Figure 4b). Changes in the SL and SE layers were not statistically significant. Thus, in conditions of complete interruption of the cerebral blood flow followed by reperfusion, demyelination occurs in the hippocampal layers adjacent directly to the CA1 field that contains the processes of pyramidal neurons, the most vulnerable to ischemia.
were found both between the control and the 10-day time point and between the control and the 30-day time point. In addition, a significant (p < 0.05) decrease in the MBP-positive area on the 30th day after ischemia was found in the SR layer compared to the control (Figure 4b). Changes in the SL and SE layers were not statistically significant. Thus, in conditions of complete interruption of the cerebral blood flow followed by reperfusion, demyelination occurs in the hippocampal layers adjacent directly to the CA1 field that contains the processes of pyramidal neurons, the most vulnerable to ischemia.  (b) comparison of myelin content between the groups according to the percentage of MBP staining area. Significant differences between the groups, according to ANOVA after LSD correction for multiple comparisons: ***-p < 0.001; **-p < 0.01; *-p < 0.05; (c) MIP images of magnified fragments of the hippocampal layers, in which significant changes in the number of MBP after GCI were revealed. 40× oil immersion objective.
The number of myelinating oligodendrocytes also in the hippocampus was reduced after GCI ( Figure 5). On Day 10 after GCI, similar to the change in myelin content, a significant decrease in the number of myelinating oligodendrocytes compared to the control was observed in the SO and SP hippocampal layers (p < 0.01). On Day 30 after GCI, the number of oligodendrocytes in the SO and SP layers was reduced both in comparison to the control (p < 0.001) and in comparison to the 10-day point (only the SP layer, p < 0.01). In addition, on Day 30, a significant reduction in the number of oligodendrocytes was observed in the SR and SL layers.
( Figure 5). On Day 10 after GCI, similar to the change in myelin content, a significant decrease in the number of myelinating oligodendrocytes compared to the control was observed in the SO and SP hippocampal layers (p < 0.01). On Day 30 after GCI, the number of oligodendrocytes in the SO and SP layers was reduced both in comparison to the control (p < 0.001) and in comparison to the 10-day point (only the SP layer, p < 0.01). In addition, on Day 30, a significant reduction in the number of oligodendrocytes was observed in the SR and SL layers. (a) General views of CNP-stained hippocampus at the CA1 field level at Days 10 and 30 after GCI and in sham-operated controls. CC-corpus callosum, SO, SP, SR, substratum lacunosum (SL) and substratum eumoleculare (SE)-hippocampal layers, DG-dentate gyrus, 20× objective; (b) comparison of CNP-positive cells between the groups. Significant differences between the groups, according to ANOVA after LSD correction for multiple comparisons: ***-p < 0.001; **-p < 0.01; (c) Magnified fragments of the hippocampal layers, in which significant changes in the number of MBP after GCI were revealed. 20× objective.

GCI Increases the Number of Immature Oligodendrocytes in the Hippocampus
In an intact brain, OPCs (NG2-positive cells) in the hippocampus have many branched, radially sprouting processes. The morphology of NG2+ cells after GCI did not change as noticeably as microglia and astrocytes activated by GCI (Figure 6c). 10 days after GCI, NG2+ cells were not as multiprocessed as in the control group and had larger cell bodies. On Day 30 after GCI on the contrary, they were distinguished by a large number of processes and, in general, looked larger, especially in the SR and SL. Quantification of the NG2+ cells showed a significantly increased number of OPCs after GCI in all hippocampal layers except the SE (Figure 6a,b). 10 days after GCI, the number of immature oligodendrocytes increased compared to control only in the SP (p < 0.001). 30 days after GCI, the number of NG2+ cells significantly exceeded both the control and the 10-day time point in the SO (p < 0.001 and p < 0.01, respectively), SP (p < 0.001), SR (p < 0.01 and p < 0.05, respectively) and SL (p < 0.001 and p < 0.01, respectively) (Figure 6b).

Discussion
Our results for the first time have demonstrated myelin and oligodendrocyte loss of the hippocampus on the rat model of GCI, which implies complete interruption of cerebral blood flow and simulates cardiac arrest in humans. We investigated the dynamics of neuronal loss, neurodegeneration, astrogliosis, inflammation, utilization of disrupted myelin by microglia/macrophages and specific rod-shaped changes of microglial morphology.

Discussion
Our results for the first time have demonstrated myelin and oligodendrocyte loss of the hippocampus on the rat model of GCI, which implies complete interruption of cerebral blood flow and simulates cardiac arrest in humans. We investigated the dynamics of neuronal loss, neurodegeneration, astrogliosis, inflammation, utilization of disrupted myelin by microglia/macrophages and specific rod-shaped changes of microglial morphology.
To the best of our knowledge, demyelination has not been studied in a rat model of GCI to date. Earlier Lee et al. [33] in the gerbil model of GCI found myelin loss in the hippocampus, which started from 4 days after ischemia. Our results show that myelin and oligodendrocyte loss was observed 10 days after GCI and intensified by the 30th day in the hippocampal layers adjacent to the pyramidal neurons in the CA1 field (SO and SR), as well as the SP layer itself. Unlike focal ischemia, when there is an interruption of the blood supply to a limited area, global cerebral ischemia leads to a temporary cessation of blood supply to the entire brain. However, short-term cessation of blood flow leads to serious damage to only certain brain regions. A distinctive feature of the global ischemia-hypoxia model in rodents is severe damage to the hippocampal pyramidal neurons in the CA1 field [13,14,31,34], which is confirmed by the results of our previous [17,19,35] and present studies. In addition, a number of studies have found damage to the neurons of the cerebellum, cortex, striatum and thalamus in global ischemia-hypoxia [36][37][38][39]. Our study also found neurodegeneration in the neocortex and thalamus, slight on the 10th day after ischemia and increasing by the 30th day. The hippocampal subfields respond differently to GCI: pyramidal neurons in the CA3 field are relatively resistant to transient global cerebral ischemia, while neurons in the CA1 field are more susceptible to hypoxia. The death of neurons in this area is called the "delayed hippocampal neuronal death" [13,14], noticeable 4-7 days after GCI in gerbils [33,40]. Lee et al. [33] found an almost complete loss of hippocampal pyramidal neurons in the CA1 field in gerbils as early as 4 days after GCI. Our results demonstrated more delayed neuronal death in the rat model of GCI: we found the death of approximately half of neurons on the 10th day and more than 90% of neurons on the 30th day after GCI the CA1 field, whereas the CA2 and CA3 fields were less affected. The higher sensitivity to ischemia of the CA1 pyramidal neurons in gerbils compared to rats may be associated with increased NO synthesis [41] or metabolic features of GABAergic pyramidal neurons in the hippocampus, which is associated with the propensity of this species to epilepsy [42,43].
Apparently, pyramidal neurons of the CA1 field die mainly by the mechanism of glutamate-dependent excitotoxicity [44], since they receive a huge number of exciting glutamate inputs, which is dozens of times more than the number of inhibitory GABA inputs [45]. Main inputs are represented by afferents from the entorhinal cortex as part of alvear and performant pathways [46], Schaffer collaterals and numerous inputs from GABAergic interneurons within the hippocampus, among which up to 12 subtypes are distinguished [47]. The hippocampal network is very complex and is still the subject of morphologic studies [47,48]. One pyramidal neuron of the CA1 field can receive more than 30 thousand inputs in different layers of the hippocampus, including the SO, SP, SR and SL [45,48]. Among these numerous inputs the long axons from entorhinal cortex within alvear and perforant are myelinated, located in all layers of the hippocampus except the SE and having contacts with the basal and apical dendrites of the CA1 pyramidal neurons [49]. In addition, the SO and SP contain the myelinated axons of the CA1 pyramidal neurons that send fibers to the subiculum, entorhinal cortex and other extrinsic brain structures [50]. The Schaffer collaterals within the SL and their branches in the SR, which contacted with apical dendrites of the CA1 pyramidal neurons, are largely unmyelinated or weakly myelinated [51].
According to our results, on Day 10 after GCI significant myelin and oligodendrocyte loss was found only in the SO and SP. By the 30th day, myelin loss in these layers intensified and affected the SR, the number of oligodendrocytes decreased in the SR and SL. It is likely that earlier changes (Day 10) were found to be associated with axonal myelin destruction of dead pyramidal neurons of the CA1 field, while later changes (30 days) also affect the demyelination of afferents from alveus and the perforant pathway in the SO, SP and SR layers.
These assumptions are confirmed by phagocytosis of myelin and the time course of the detected morphologic changes in glial cells in the above layers of the hippocampus. In addition to the expected activation and increase in the number of astrocytes and microglia in these layers, which is consistent with numerous studies [52][53][54], we observed specific morphologic changes in these cells in the SR on the 10th day after GCI, associated with the orientation and extension of their bodies parallel to myelinated fibers and perpendicular to the layer of pyramidal neurons.
A morphology similar to that which we observed in the SR 10 days after ischemia-an elongated rod-shaped nucleus and a body with short processes, is characteristic of rod-shaped microglia [55][56][57][58]. Rod-shaped microglia have been described in the cortex in rats with diffuse brain injury [56], in the hippocampus in older adults [58] and present in experimental models of optic nerve degeneration and in some slowly developing neurodegenerative diseases progressing to dementia [55]. Individual cells or chains from rod microglia are adjacent to neuronal processes. It was shown that postischemic changes in the SR layer of the hippocampus are limited to the region of synaptic terminals (clumping or dispersion of the synaptic vesicle pools and damage to synaptic membranes) and synaptic terminals are the primary and early target in the development of damage to the postischemic neurons [14,26]. It is suggested that a close relationship with the processes of neurons reflects the participation of Rod microglia in synaptic stripping and neuronal circuitry reorganization [56,59]. The study of microglia-synaptic interactions showed that there is not complete destruction of the synapses, but selective partial phagocytosis or trogocytosis, of presynaptic structures [60]. The cell bodies of rod microglia, which we observed in the SR 10 days after GCI, spread along myelinated axons and made contact with them by their short processes. We suggest that together with the utilization of myelin and other cellular components, the rod microglia in this layer "disconnects" the excitatory inputs of pyramidal neurons from the afferents of the alvear and performant pathways to prevent neuronal loss due to glutamate excitotoxicity. With continued neuroinflammation on the 30th day after GCI, an accumulation of activated microglia, mainly with large round cell bodies and short processes, was observed in the SR and the phagocytosis occurs more actively. This corresponds to a further decrease in the MBP-positive area in the SR at Day 30 after GCI.
An interesting observation was the appearance of different types of contacts of rod-shaped microglia with myelinated axons including contacts by the bipolar ends of microglial bodies, by the microglial lateral processes and close grasping contact of microglial bodies with myelin fibers. Recent studies reveal that microglia and astrocytes play a major role in network formation during early development, in adulthood and neurodegeneration by directly pruning redundant synapses [61]. Microglia are also able to act as a sensor of synaptic activity and play a key role in the homeostatic regulation of neural excitation [62][63][64]. We assume that the bipolar contacts of rod-microglia bodies with myelinated fibers that we found, as well as the contacts of the lateral processes in the nodes of Ranvier, act as sensors for searching for synapses with excessive excitation and for pruning of such a contact. Similar pruning of superfluous excitatory synapses is shown during neurodevelopment [65]. Verification of these assumptions, as well as the functional significance of the third type of contacts, which is a close grasping contact of microglial bodies with myelin fibers, is the subject of further research.
According to our results, in parallel with ongoing myelin and oligodendrocyte loss on the 30th day after ischemia, remyelination processes begin, as evidenced by an increase in the number of OPCs in the hippocampus. A significant increase in the number of OPCs in the SO, SP and SR layers of the hippocampus, the most subjected to demyelination after GCI, indicates the beginning of the regenerative process of remyelination. The number of OPCs in these layers significantly increases by the 10th day and multiplicatively increases on the 30th day after GCI. NG2+ glia is recognized as a separate glial cell population giving rise to oligodendrocytes [66]. In response to damage, NG2+ glial cells are not only able to proliferate and migrate to lesions, but also differentiate into oligodendrocytes, forming new myelin sheaths that wrap around damaged axons and lead to their functional recovery [67,68].
In addition, the results of genomic and epigenetic studies have shown that reactive NG2 glia can also differentiate into GFAP-labeled astrocytes and DCX-expressing immature neurons after traumatic injury [69][70][71].

Animals and Housing
The study was performed on adult male Wistar rats weighing 250-300 g (n = 29; 15 rats survived after surgery) obtained from the vivarium of the E.D. Goldberg Institute of Pharmacology and Regenerative Medicine, Tomsk, Russia. Experiments were carried out in accordance with the rules adopted by the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The study was approved on 22 March 2012 by the Animal Care and Use Committee at the E.D. Goldberg Institute of Pharmacology and Regenerative Medicine (protocol #22032012). Animals were housed in groups of seven animals per cage with about 300 cm 2 per animal under standard conditions (12/12-h light/dark cycle, temperature of 22 ± 2 • C, humidity of 60%). Standard rodent chow (PK-120-1, Laboratorsnab, Ltd., Moscow, Russia) and water were provided ad libitum.

Experimental Design
Animals were randomly divided into two groups: sham-operated animals (n = 5) and animals, which underwent GCI (n = 10). Acute global cerebral ischemia was induced according to the new three-vessel model [16].
The surgery was performed as described previously [16,72]. Briefly, rats were anesthetized with chloral hydrate in a dose of 450 mg/kg intraperitoneally (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and placed on a homeothermic blanket (Temperature Control Unit HB 101/2, Letica Scientific Instruments, Barcelona, Spain) in a supine position. The body temperature was maintained at 37 • C. Cerebral blood flow was completely interrupted for 7 min by ligation of the major arteries supplying the brain (truncus brachiocephalicus, arteria subclavia sinistra, and arteria carotis communis sinistra). Access to arteria carotis communis sinistra was implemented through the ventral surface of the neck, while truncus brachiocephalicus and arteria subclavia sinistra were reached through the first intercostal space, bypassing the pleural cavity to avoid pneumothorax. The animals were intubated through the oral cavity. The same surgery was performed in sham-operated rats, but without ligation of blood vessels.
At Days 11 and 31 after surgery, neurological deficit was evaluated with the Stroke-index McGraw scale [72,73].
Animals were euthanized at Days 11 and 31 after surgery by transcardial perfusion with 4% paraformaldehyde under ether anesthesia. The brains were removed, fixed overnight with 4% paraformaldehyde solution, cryoprotected in sucrose phosphate buffer (24 h in 10% and 24 h in 20% solutions, respectively) at 4 • C, frozen in liquid nitrogen and stored at −80 • C for further immunohistochemical studies.

Immunochemistry and Microscopy
Coronal brain sections with 10-µm thickness were prepared using an HM525 cryostat (Thermo Fisher Scientific, Walldorf, Germany). Brain locations for immunohistochemical analysis were defined from −2.64 mm to −3.60 mm from bregma according to a rat brain atlas [74].
The following primary antibodies were used: Brain sections were incubated with primary antibodies carried out overnight at 4 • C, with secondary antibodies-for 3 h at RT. Afterwards, stained brain sections were covered with antifade mounting medium Vectashield with DAPI (40,6-diamidino-2-phenylindole).
For each animal at least 4 microphotographs of the whole hippocampus of both (the left and right) hemispheres were obtained. Hippocampus general plans were photographed using an Axio Imager.Z2 (Carl Zeiss, Oberkochen, Germany) microscope (objective lens Plan-Apochromat 20×) and AxioVision 4.8 (Carl Zeiss) software with a MozaiX program module. 3D micrographs of separate groups of cells were obtained using Axio Imager Z1 (Carl Zeiss, Oberkochen, Germany) microscope (objective lens EC Plan-Neofluar 40× oil) with 3D ApoTome imaging system as a set of Z-stacks (45-50 sections per image), followed by the creation of a maximum intensity projection (MIP) image and 3D cell models.

Image Processing
Calculation of NeuN+, GFAP+, Iba1+, NG2+, CNP+ cells was carried out using ImageJ software (National Institutes of Health, Bethesda, MD, USA). To quantify the severity of the ischemic lesion, cell count change in CA1 field of the hippocampus was evaluated by visual calculation of cells in the stratum oriens (SO), stratum pyramidale (SP), substratum radiatum (SR), substratum lacunosum (SL) and substratum eumoleculare (SE). Double-labeled FJC+\NeuN+ cells were calculated in the SP layer. The cells were counted within regions of interest (ROIs) according to the scheme presented in Figure 7. Three ROIs pre section, from 4 to 5 sections of the left and right hemispheres for each animal were analyzed (ROI: SO, SL, SE-500 × 250; SP-500 × 100; SR-500 × 500). Calculated number of cells per ROI were normalized to 1 mm 2 area.
Myelin content in the hippocampal layers was assessed in ROIs similar to those for calculation of NeuN+, GFAP+, Iba1+, NG2+, CNP+ cells in the layers SO, SP, SR, SL and SE near the CA1 field ( Figure 6). Assessment was performed using the Otsu thresholding method in the ImageJ (National Institutes of Health, Bethesda, MD, USA) implementation as a percent of MBP-positive area [35,75,76].
Myelin content in the hippocampal layers was assessed in ROIs similar to those for calculation of NeuN+, GFAP+, Iba1+, NG2+, CNP+ cells in the layers SO, SP, SR, SL and SE near the CA1 field ( Figure  6). Assessment was performed using the Otsu thresholding method in the ImageJ (National Institutes of Health, Bethesda, MD, USA) implementation as a percent of MBP-positive area [35,75,76].

Statistical Analysis
Statistical analysis was performed using the Statistica 10.0 software (StatSoft, Inc., Tulsa, OK, USA). Mean values and standard errors of mean (SEM) for each type of labeled cells were calculated in the hippocampal layers around the СА1 fields: SO, SP, SR, SL and SE. Quantitative histology data were compared between the control, 10-day ischemic and 30-day ischemic groups using a two-way repeated measures analysis of variance (ANOVA) followed by post hoc least significant difference (LSD) tests for individual layers of the hippocampus. The statistical significance for all the analyses was less than 0.05.

Statistical Analysis
Statistical analysis was performed using the Statistica 10.0 software (StatSoft, Inc., Tulsa, OK, USA). Mean values and standard errors of mean (SEM) for each type of labeled cells were calculated in the hippocampal layers around the CA1 fields: SO, SP, SR, SL and SE. Quantitative histology data were compared between the control, 10-day ischemic and 30-day ischemic groups using a two-way repeated measures analysis of variance (ANOVA) followed by post hoc least significant difference (LSD) tests for individual layers of the hippocampus. The statistical significance for all the analyses was less than 0.05.

Conclusions
Our study showed that GCI caused by a complete 7-min interruption of cerebral blood flow is accompanied not only by the loss of pyramidal neurons in the CA1 field, neurodegeneration of neocortex and thalamus, astrogliosis and inflammation in SP and adjacent layers of SO and SR, but also by myelin and oligodendrocyte loss in these layers of the hippocampus. At an earlier time-period postischemia (10 days), significant myelin and oligodendrocyte loss is observed in the SO and SP layers, and by 30 days it affects the SR layer, in which, presumably, an activated rod-like microglia first disconnects the contacts of the dying neurons and then utilizes myelin, axons, neurons and other cellular components. In parallel with the pathologic processes of neuronal death, demyelination and ongoing inflammation in the SO, SP and SR layers, the signs of restoration in the affected area of the hippocampus appear -a significant increase in the number of OPCs (NG2-positive cells) in the layers affected by GCI. Future long-term studies are needed to clarify the timeline of destruction and the degree of functional recovery of the hippocampal neural network after global brain ischemia that accompanies cardiac arrest. Another area of research may be related to elucidation of the functional role of rod-shaped microglia in the SR after GCI, which, probably, pruning excitatory synaptic contacts and remodel the hippocampal neural network to reduce neuronal loss.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/17/ 6246/s1. Figure S1: Neurodegeneration in the neocortex 10 and 30 days after GCI. Figure S2: Neurodegeneration in the thalamus 10 and 30 days after GCI. Acknowledgments: The authors would like to acknowledge Anna Naumova for revising the final version of the manuscript and her helpful comments. The authors thank Anton Kobrin for proofreading the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.