Histological and immunohistochemical study of experimentally induced concussion on young rats’ frontal cortex and the possible protective role of erythropoietin hormone supplementation : Egyptian Journal of Histology

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Histological and immunohistochemical study of experimentally induced concussion on young rats’ frontal cortex and the possible protective role of erythropoietin hormone supplementation

Kallini, Dalia F.; El-Beshbishy, Rana A.

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The Egyptian Journal of Histology 36(3):p 611-624, September 2013. | DOI: 10.1097/01.EHX.0000433264.53866.e9
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Abstract

Introduction

The frontal lobe of the human brain is both relatively large in mass and less restricted in movement than the posterior portion of the brain. Because of its location in the anterior part of the head, the frontal lobe is arguably more susceptible to injuries 1.

Traumatic brain injury (TBI), a leading cause of death and disability, results from an outside force that initiates two phases: a primary phase, with immediate mechanical disruption of the brain tissue, and a secondary phase, with delayed pathophysiological changes that contribute toward a widespread neurodegeneration 2. Closed-head concussive injury is one of the most common causes of TBI. Although a single concussion results in short-term neurologic dysfunction, it is not known whether the concussed brain is structurally damaged as in other types of brain injury or it mainly involves physiological loss of function without structural changes. Recently, it was believed that both structural and psychiatric factors are responsible for the effects of concussion. Multiple concussions can result in cumulative damage and increased risk for neurodegenerative disease in later life, such as dementia, Parkinson’s disease, and/or depression. Children are more prone to repeated head trauma mostly because of falls or motor vehicle accidents 3. Despite the prevalence of concussion, knowledge of the sequences that occur in the brain following this injury is limited, to some extent because of the limited number of appropriate animal research models 4,5.

At present, there are no therapeutic interventions capable of counteracting or repairing TBI-induced brain damage, despite extensive research 6. As the immediate cell death, resulting from the initial impact on the brain tissue, is irreversible, treatments focus on interruption or inhibition of the secondary injury cascades that expand this primary injury. Nonetheless, no effective neuroprotective treatment is available so far 7.

Erythropoietin (EPO) is a hematopoietic cytokine produced by the perisinusoidal cells in the fetal liver and by the peritubular capillaries endothelial cells in the adult kidney in response to hypoxia 8. Recent studies have clarified its incorporation into other biological functions, in addition to its crucial hormonal role in red cell production; it functions as a tissue-protective cytokine 9. The expression of EPO and its receptors in the central nervous system (CNS) together with its upregulation by hypoxia/ischemia both in vivo and in vitro suggest the important role of this cytokine in the brain’s response to ischemic damage. Consistent with this suggestion, pretreatment with exogenous EPO protects cultured neurons from hypoxia; EPO is also involved in the wound-healing process. When administered systemically, EPO can cross the blood–brain barrier and reduce neuronal injury in animal models of focal ischemic stroke and TBI 10. Moreover, interestingly, the expression of the EPO and its receptor was observed early in fetal development in the human brain 11. Thus, the aim of the present study was to mimic a repeated brain concussion model in immature rats to clarify its effect on the architecture of the frontal cortex and to determine the effect of supplementation of exogenous EPO on the post-traumatic brain outcome.

Materials and methods

Twenty male albino rats, 17–19 days old, weighing 40–50 g, were purchased from the Research Unit and Bilharzial Research Center of Faculty of Medicine, Ain Shams University (Cairo, Egypt). The animals were kept in adequate ventilation and temperature in plastic cages, with 12-h light : 12-h dark cycles, and were fed standard laboratory food and water ad libitum. The rats were divided into four groups:

Group I: the control group, included five albino rats that were not subjected to any procedure.

In the experimental groups, 15 albino rats were subjected to repeated concussions using the weight-drop model, which was considered the original TBI model described previously 12. The rats were immobilized and then a focal impact was produced by a freefalling weight of 100 g guided in a tube that was made to hit the exposed skull in the middle line anterior to the coronal suture from a height of 25 cm 13. This impact produced a concussive-like TBI effect 14,15 and following trauma, the rats underwent a transient period of loss of consciousness, followed by tremors, limb spasticity, and convulsions that lasted from a few seconds to a few minutes. The rats were then left to recover the rest of the day. The procedure was repeated daily for 3 successive days. About 30% of rats did not withstand the repeated concussions and died and were replaced by others.

This methodology was approved by the Animal Care and Use Committee of Ain Shams University.

These rats subjected to concussion were then subdivided equally into three groups:

Group II: the concussion group, included animals that were sacrificed 24 h after the last concussion.

Group III: the recovery group, included animals that were left to recover without treatment for 10 days after the last concussion and then sacrificed.

Group IV: the treated group, included animals that were injected by an injectable recombinant human EPO that was provided by the Egyptian Pharmex Company (10 000 IU/1 ml) Shenyang Sunshine Pharmaceutical Co., China. EPO was injected intraperitoneally at a dose of 5000 U/kg body weight starting within the first 6 h after the trauma for 3 consecutive days 16. The animals were left for 1 week and then sacrificed.

All animals were sacrificed with a lethal dose of ether according to the protocol of the Animal Care and Use Committee of Ain Shams University. The skulls were opened. The brains were exposed and then carefully and completely extracted from the cranial cavity. In each specimen, one hemisphere was fixed immediately in a 10% neutral-buffered formalin solution and processed for light microscopic examination. In the other hemisphere, the frontal cortex was identified, cut into small pieces, placed in cold buffered gluteraldehyde 3.5%, and then processed for electron microscopy examination.

Light microscopic study

Specimens, after being fixed in 10% neutral-buffered formalin for 1 week, were dehydrated, cleared, and embedded in paraffin wax. Sections (5-μm thick) were cut and stained with H&E. Other specimens fixed in cold buffered gluteraldehyde 3.5% were processed to Epon blocks, trimmed, and cut into semithin sections (1-μm thick) using an LKB microtome. The semithin sections were stained with toluidine blue 17. The sections were examined using an Olympus light microscope and photographed.

Immunohistochemical study for glial fibrillar acid protein

Glial fibrillar acid protein (GFAP) immunostaining is the most commonly used method to examine the distribution of astrocytes and their response to neural degeneration or injury. The modified Avidin–biotin immunoperoxidase technique for GFAP was applied to demonstrate astrocytes 18. Primary anti-GFAP antibody Goat polyclonal IgG, anti-rat, produced by Dako Cytomation, was used. Working dilution was 1:1000. An immunohistochemical reaction was carried out using the avidin–biotin–peroxidase system, followed by incubation with 1/100 normal rabbit serum for 20 min in order to exclude nonspecific background. GFAP-containing cells (astrocytes) appeared brown and nuclei appeared blue because of counterstaining with hematoxylin 17.

Immunohistochemical study for synaptophysin

Synaptophysin (SYN) is a synaptic vesicle glycoprotein. It is present in neuroendocrine cells and in almost all neurons in the brain and spinal cord that participate in synaptic transmission. It acts as a marker for neuroendocrine tumors, and its ubiquity at the synapse has led to the use of SYN immunostaining for the quantification of synapses 19.

Deparaffinized sections were rinsed three times, 10 min each, in PBS (0.1 mol/l sodium phosphate buffer in 0.9% saline; pH 7.4). Sections were blocked with 5% normal horse serum (NHS; Vector Laboratories, Burlington, Ontario, Canada) and 3% Triton-X (Boehringer Mannheim, Laval, Quebec, Canada) for 15 min and then incubated for 2 h in mouse monoclonal antibody against SYN (1:200; Sigma-Aldrich Chemicals) at room temperature. Sections were then incubated in mouse secondary antisera (1:200; Vector Laboratories) for 2 h at room temperature. Sections were then incubated in avidin–biotin–horseradish peroxidase complex (1:50, ABC Elite Kit; Vector Laboratories) for 90 min. Sections were reacted in 0.01% diaminobenzidine (DAB; Sigma-Aldrich Chemicals) with 0.0003% H2O2 for ∼5 min. The sections were mounted on super-frost slides, dried overnight, dehydrated, and then cover slipped 17.

Transmission electron microscopy

Specimens with a volume of about 1.0 mm3 were washed twice with PBS and fixed in a mixture of 1% gluteraldehyde and 2% paraformaldehyde in phosphate-buffer at room temperature (pH 7.4) for 24 h. The specimens were then washed twice in buffered sucrose for 5 min. Each specimen (0.1 mol/l phosphate buffer, 5% sucrose solution) was then postfixed in phosphate-buffered 2% osmium tetroxide at 4°C for 60 min. The specimens were dehydrated in ascending grades of ethanol and embedded in epoxy resin. Ultrathin sections were cut with glass knives on an ultratome, stained with uranyl acetate and lead citrate, followed by carbon coating. The specimens were examined and photographed using a transmission electron microscope (TEM-100 SEO) 20.

Morphometric study

The photomicrograph of each section was taken using an Olympus E-330 Live View Digital SLR Camera in the Department of Anatomy, Ain Shams University. The same area was selected in the frontal cortex in all sections. To measure the density of pyramidal/degenerated cells in an area, the photos were transferred to the computer. The appropriate grids were superimposed on the photos and the cells were counted by two independent observers blinded to the specimen details to perform an unbiased assessment 21.

Quantitative image analysis

The measurements were performed using the image analyzer (Leica Q 500 M C program) in the Faculty of Medicine, Ain Shams University. The image analyzer consists of a colored video camera, a colored monitor, and an IBM personal computer connected to the microscope. The image analyzer was first calibrated automatically to convert the measurement units (pixels) produced by the image analyzer program into actual micrometer units. Each field was enclosed inside the standard measuring frame and then the positively reacting GFAP and SYN immune-stained areas were masked by a blue binary color to be measured. The density (area percentage) of the immune staining was measured in five fields from five serial sections from five animals from each group 22.

Statistical analysis

Numerical variables were summarized by means and SD; the analysis of variance test was used for comparison between the four groups studied for quantitative data, number of degenerated cells, intensity of GFAP, and SYN reactions. Fisher’s least significant difference test was used as a post-hoc test. SPSS, version 15 was used for statistical analysis. The significance of data was determined by the P value (P≤0.05 was considered significant and P≤0.001 was considered highly significant).

Results

The frontal cortex of the control group (group I) was covered by pia mater and loosely stratified into six layers containing scattered nuclei of both neurons and glial cells. These layers were identified as the outer molecular layer (I), external granular layer (II), external pyramidal layer (III), internal granular layer (IV), internal pyramidal layer (V), and polymorphic layer (VI) (Fig. 1). Neurons of the frontal cortex were of varying shapes and sizes, but the most obvious were the pyramidal cells. Each pyramidal cell in the external pyramidal layer appeared triangular in shape with a basophilic cytoplasm and a centrally located large vesicular nucleus (Figs 2 and 3). The smaller neuroglia cells, few granular cells, and blood capillaries were scattered in the neuropil (Fig. 2). Immunohistochemical staining for GFAP in the control group was only positive for the astrocytes. The reaction was diffuse, evenly distributed, and more pronounced adjacent to the blood vessels (Fig. 4). The cytoplasm of the astrocytes together with their processes showed a positive reaction for GFAP that appeared brown in color. Few astrocytes were observed in between the pyramidal cells with a bushy or a starry appearance (Fig. 5). Positive SYN immunoreactivity was observed uniformly distributed outlining the neurons within the neuropil. This reaction was absent in the neurons and blood vessels (Fig. 6). Ultrastructurally, the nucleus of the pyramidal neurons appeared rounded, and euchromatic, with a prominent nucleolus. The perikaryal cytoplasm showed organelles typical of pyramidal cells (Fig. 7).

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Figure 1:
A photomicrograph of a section in the frontal cortex of a control rat (group I) showing the motor cortex covered by pia mater (→). The six layers of the frontal cortex can be identified: outer molecular layer (I), external granular layer (II), external pyramidal layer (III), internal granular layer (IV), internal pyramidal layer (V), and polymorphic layer (VI). H&E, ×100.
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Figure 2:
A photomicrograph of a section in layer III of the frontal cortex of a control rat (group I) showing pyramidal cells (P) with rounded/oval vesicular nuclei, basophilic cytoplasm, and processes. Note the neuroglia cells (white arrow), granular cells (→), and blood vessels (arrow heads) in the neuropil (*). H&E, ×400.
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Figure 3:
A photomicrograph of a section in layer III of the frontal cortex of a control rat (group I) showing the pyramidal cells (→) with large vesicular nuclei and darkly stained cytoplasm. Toluidine blue, ×1000.
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Figure 4:
A photomicrograph of a section in the frontal cortex of a control rat (group I) showing the distribution of glial fibrillar acid protein (GFAP) immunoreactivity across the six cortical layers. Note the GFAP immunoreactivity more adjacent to blood vessels. Immunoperoxidase technique for GFAP, ×100.
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Figure 5:
A photomicrograph of a section in layer III of the frontal cortex of a control rat (group I) showing positive immunoreactivity for glial fibrillar acid protein (GFAP) in the cytoplasm of astrocytes and its processes (→) producing a bushy or a starry appearance. Immunoperoxidase technique for GFAP, ×400.
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Figure 6:
A photomicrograph of a section in layer III of the frontal cortex of a control rat (group I) showing positive synaptophysin (SYN) immunoreactivity distributed diffusely within the neuropil (arrow head). Note the neurons (arrow) and blood vessels (white arrow). Avidin–biotin–peroxidase for SYN, ×400.
F7-11
Figure 7:
A transmission electron micrograph of a pyramidal cell in layer III of a control rat frontal cortex (group I) showing a euchromatic rounded nucleus (N) with a prominent nucleolus (arrow head). The perikaryal cytoplasm contains many mitochondria (→). Uranyl acetate, ×8000.

The pia mater of the frontal cortex of the concussion group (group II) was raised by marked subpial inflammatory cellular infiltration and numerous congested blood vessels. The focal injury extended in the superficial layers of the frontal cortex, leading to loss of its architecture (Fig. 8). There were scanty unaffected pyramidal cells. The marked decrease in the number of normal pyramidal cells was replaced by numerous degenerated peculiar-shaped neurons, with deeply stained acidophilic cytoplasm and ill-defined darkly stained nuclei with an increase in perineuronal space (Figs 9 and 10). The increase in the number of these degenerated neurons was found to be statistically highly significant compared with the control group (Table 1). Immunohistochemically, the frontal cortex of rats of the concussion group showed a marked reduction of GFAP across the six cortical layers compared with the control group (Fig. 11). The astrocyte immunoreactivity for GFAP was highly reduced (Fig. 12). This reduction in the GFAP intensity was found to be highly statistically significant compared with the control group (Table 1). The neuropil showed markedly decreased SYN immunoreactivity. However, faint areas of reactivity were observed around small blood vessels (Fig. 13). The intensity of the SYN reaction of this group was significantly decreased compared with the control group (Table 1). Examination of ultrathin sections of the external pyramidal layer of this group showed marked degenerative changes in almost all neurons. The pyramidal cells were dark, degenerated, and peculiarly shaped. Their nuclei were dense, hyperchromatic, and fragmented. The surrounding neuropil appeared markedly vacuolated (Fig. 14).

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Figure 8:
A photomicrograph of a section in the frontal cortex of a rat from the concussion group (group II) showing subpial (*) inflammatory cells’ infiltration and numerous congested blood vessels (→). Note the loss of frontal cortex architecture (thick arrow) and the most affected region is the superficial layers (bracket). H&E, ×100.
F9-11
Figure 9:
A photomicrograph of a section in layer III of the frontal cortex of a rat from the concussion group (group II) showing numerous degenerated neurons (→) with deeply stained acidophilic cytoplasm and ill-defined nuclei with an increase in the perineuronal space. Some cells appear unaffected (white arrow) within the neuropil (*). Note the blood vessels (arrow heads). H&E, ×400.
F10-11
Figure 10:
A photomicrograph of a section in layer III of the frontal cortex of a rat from the concussion group (group II) showing an apparent decrease in the number of pyramidal cells. The neurons appear peculiarly shaped (→), with darkly stained nuclei. Toluidine blue, ×1000.
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Table 1:
Image analysis of number of degenerated cells, and intensity of glial fibrillar acid protein and synaptophysin reactions in 400 HPF
F11-11
Figure 11:
A photomicrograph of a section in the frontal cortex of a rat from the concussion group (group II) showing the distribution of glial fibrillar acid protein (GFAP) immunoreactivity across the six cortical layers. Note the decreased reactivity. Immunoperoxidase technique for GFAP, ×100.
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Figure 12:
A higher magnification of a section in layer III in the frontal cortex of a rat from the concussion group (group II) showing decreased immunoreactivity for glial fibrillar acid protein (GFAP). Astrocytes (→). Immunoperoxidase technique for GFAP, ×400.
F13-11
Figure 13:
A photomicrograph of a section in layer III in the frontal cortex of a rat from the concussion group (group II) showing decreased synaptophysin (SYN) immunoreactivity within the neuropil (arrow heads). Note neurons (white arrows) and blood vessels (→). Immunoperoxidase technique for SYN, ×400.
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Figure 14:
A transmission electron micrograph in layer III in the frontal cortex of a rat from the concussion group (group II) showing two dark peculiar-shaped degenerated neurons (red arrows). Their nuclei appear dense hyperchromatic and fragmented. Vacuolation (*) could be identified in the adjacent neuropil. Uranyl acetate, ×12 000.

The subpial space of the motor cortex of the recovery group (group III) was reduced compared with the concussion group. The cortical layers showed marked disorganization and hypercellularity, with a marked decrease in the typical neuronal appearance (Fig. 15). Some cells were still affected; they appeared deeply stained, with ill-defined nuclei and an increase in the perineuronal space (Fig. 16), whereas others appeared regenerated with a normal pyramidal appearance (Fig. 17). Although the number of degenerated pyramidal cells was less than that of the concussion group, there was still a statistically highly significant difference compared with the control group (Table 1). Immunohistochemically, the evenly distributed immunostaining of GFAP was disrupted across the six cortical layers, where it was more pronounced in the deeper layers of the cortex (Fig. 18). In the third layer, an increase in GFAP immunoreactivity was observed in the hypertrophied astrocytes and their numerous processes (Fig. 19). A statistically highly significant increase in the GFAP immunoreactivity was observed in this group than both the control and the concussion groups (Table 1). However, the positive SYN immunoreactivity within the neuropil was increased in comparison with the concussion group (Fig. 20). The increase in the SYN immunoreactivity was statistically highly significant compared with both the control and concussion groups (Table 1). Ultrastructurally, most of the pyramidal cells in the third layer of the frontal cortex of the rats in the recovery group showed nuclei with a normal appearance. Some vacuoles could be observed enclosed in the cytoplasm of the pyramidal cells and within the surrounding neuropil (Fig. 21).

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Figure 15:
A photomicrograph of a section in the frontal cortex of a rat from the recovery group (group III) showing the pia (arrow heads) and decreased subpial space. Note disorganization and hypercellularity of the cortical layer (white arrows) and congested blood vessels (thick arrow). H&E, ×100.
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Figure 16:
A photomicrograph of a section in the frontal cortex of a rat from the recovery group (group III) showing hypercellularity of the cortex. Some cells are deeply stained, with ill-defined nuclei (thick arrows) and an increase in the perineuronal space. H&E, ×400.
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Figure 17:
A photomicrograph of a section in layer III of the frontal cortex of a rat of the recovery group (group III) showing normal appearance of some pyramidal cells (→). Some darkly stained neurons (arrow heads) can be seen. Toluidine blue, ×1000.
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Figure 18:
A photomicrograph of a section in the frontal cortex of a rat of the recovery group (group III) showing glial fibrillar acid protein (GFAP)-positive immunoreactivity in the cortical layers that is clearly more pronounced in the deeper layers of the cortex. Immunoperoxidase technique for GFAP, ×100.
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Figure 19:
A photomicrograph of a section in layer III of the frontal cortex of a rat of the recovery group (group III) showing an increase in glial fibrillar acid protein (GFAP) immunoreactivity in hypertrophied astrocytes (→) and their numerous processes. Immunoperoxidase technique for GFAP, ×400.
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Figure 20:
A photomicrograph of a section in layer III of the frontal cortex of a rat of the recovery group (group III) showing an increase in synaptophysin (SYN) immunoreactivity. Immunoperoxidase technique for SYN, ×400.
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Figure 21:
A transmission electron micrograph of a pyramidal cell (red arrow) in layer III of the frontal cortex of a rat of recovery group (group III) showing normal appearance of the nucleus (N). The cytoplasm shows some vacuoles (thin arrows). The surrounding neuropil appears vacuolated (*). Uranyl acetate, ×10 000.

In H&E-stained sections of the treated group (group IV), the injection of EPO for 3 days led to good recovery as the frontal cortex apparently regained its normal appearance, with reappearance of almost normal neurons (Figs 22 and 23). The cellular infiltration and subpial space, observed previously, showed marked improvement (Fig. 22). The pyramidal cells restored their normal appearance, where almost all of the cells showed vesicular nuclei. The deeply stained nuclei of adjacent neuroglia were apparent (Fig. 23). Also, in semithin sections, many pyramidal cells restored their large vesicular nuclei. However, few of them were apparently still affected. They appeared as darkly stained cells (Fig. 24). The number of degenerated pyramidal cells was comparable to that in the control group; however, it was statistically significantly less than the concussion and recovery groups (Table 1). GFAP-stained sections of this group showed a normal distribution of immunoreactivity across the six cortical layers, comparable to the control group (Figs 25 and 26). Although the number of astrocytes and the intensity of GFAP immunoreactivity in their small body and their thick cell processes were evenly distributed and apparently similar to that of the control group, a statistically significant difference was found between the two groups (Fig. 26 and Table 1). Also, a highly significant difference was found between the GFAP immunoreactivity in the treated group and both the concussion and recovery groups (Table 1). SYN-stained sections of the frontal cortex of the rats of the treated group showed positive immunoreactivity almost comparable to that of the control group (Fig. 27); however, they were statistically significantly higher than those of the concussion and recovery groups (Table 1). Ultrastructurally, most of the pyramidal cells in the third cortical layer of the motor cortex had recovered. Their nuclei regained their rounded or oval shape, their euchromatic appearance, and prominent nucleoli. The cytoplasm appeared granular because of the presence of free ribosomes, with apparent mitochondria (Fig. 28).

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Figure 22:
A photomicrograph of a section in the frontal cortex of a rat of the treated group (group IV) showing normal appearance of the six layers of the frontal cortex: outer molecular layer (I), external granular layer (II), external pyramidal layer (III), internal granular layer (IV), internal pyramidal layer (V), and polymorphic layer (VI). Note the pia (→) and subpial space. H&E, ×100.
F23-11
Figure 23:
A photomicrograph of a section in the frontal cortex of a rat of the treated group (group IV) showing normal appearance of pyramidal cells (P). Almost all the cells show vesicular nuclei. The deeply stained nuclei of adjacent neuroglia are apparent (white arrows). H&E, ×400.
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Figure 24:
A photomicrograph of a section in layer III in the frontal cortex of a rat of the treated group (group IV) showing pyramidal cells (→) with large vesicular nuclei. Few darkly stained cells appear in the section (arrow heads). Toluidine blue, ×1000.
F25-11
Figure 25:
A photomicrograph of a section in the frontal cortex of a rat of the treated group (group IV) showing normal distribution of glial fibrillar acid protein (GFAP) immunoreactivity across the six cortical layers. Immunoperoxidase technique for GFAP, ×100.
F26-11
Figure 26:
A higher magnification of a section in layer III in the frontal cortex of a rat of the treated group (group IV) showing positive immunoreactivity for glial fibrillar acid protein (GFAP). Note the reactive astrocytes (→) with darkly labeled small body and thick cell processes. Immunoperoxidase technique for GFAP, ×400.
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Figure 27:
A photomicrograph of a section in layer III in the frontal cortex of a rat of the treated group (group IV) showing positive synaptophysin (SYN) immunoreactivity. Immunoperoxidase technique for SYN, ×400.
F28-11
Figure 28:
A transmission electron micrograph in layer III in the frontal cortex of a rat of the treated group (group IV) showing two pyramidal cells with their rounded/oval euchromatic nuclei (N) and prominent nucleoli. The cytoplasm appears granular and shows mitochondria (arrows). Uranyl acetate, ×10 000.

Discussion

In this study, 17–19-day-old rats were used; this age is neurologically equivalent to a human child between the ages of 1 and 3 years 23. Higher morbidity and mortality were recorded in two risk groups: the first group was 0–4 years old and the second group was 15–19 years old 3. During the first 3 years of life, children show completely different neurotraumatic pathology when compared with adults 24.

The concussive-like brain injury model was easier to study at a young age because of the thinner skull bones 25 and was previously used in rodents of the same weight and height 14. This technique is quick, easy, and convenient as no surgery is required, hence its popularity. Transient neurobehavioral depression, short duration of brain edema, and long-lasting memory deficits have been reported previously as characteristics of this model 14. The present study investigated the early and late histopathological changes accompanying the repeated concussions in this concussive-like brain injury model to correlate them with previous recorded signs.

Repeated concussions handled in the current work were also manipulated daily by other authors for 4 days in mice. Subsequent neurological affection and neurocognitive function deficits were recorded in repeated concussions more than that in the single concussion group. Accordingly, repeated head concussions in animal models can reproduce some of the deficits observed after repeated concussions in humans and may be suitable for the discovery of novel therapies 6.

To elucidate the early and late effects of concussion, 1 day and 10 days following trauma were selected in this study, respectively. Previous studies 13,26 examined the consequences of concussive brain injury after 3, 7, and 14 days. Another study investigated the same days in immature rats using different levels of severity. On concussion with 100 g/2 m, only a motor deficit was recorded. It was severe at 24 h and persisted for 3–4 days. However, on trauma with 150 g/2 m, the resultant motor deficit was marked, reversible, and lasted for 10 days, and a sustained cognitive dysfunction was observed for 22 days 27.

The frontal cortex of the current investigation showed subpial inflammatory cellular infiltration and numerous congested blood vessels 24 h after trauma. The focal injury led to cellular infiltration, edema, and degenerated neurons. The first reaction to CNS injury is typically migration of macrophages and local microglia to the damaged site 28,29. Previously, it was reported that failure of CNS regeneration after TBI can be partly attributed to the inhibitory environment created at the site of the lesion. The glial scar formation, consisting predominantly of astrocytes, forms both a physical and a physiological barrier for axon regeneration 28.

The light and electron microscopic results of the current work observed 24 h following concussion indicated all signs of severe neuronal affection. The neurons with pyknotic nuclei and vacuolated neuropil are indicative of acute neuronal death, and are consistent with neuronal necrosis as seen in the early stages of ischemic, hypoxic/ischemic, hypoglycemic, and excitotoxic states 30. These lesions were markedly apparent in the cerebral cortex, especially in the third layer 31. Also, recent studies reported that neurons in the superficial layers of the motor cortex were the most affected, showing signs of chromatolysis, chromatin clumping, deeply stained nuclei, and pyknosis indicating neuronal degeneration. Vacuolation of neuropil and cytoplasm of pyramidal cells and the presence of widening of perineuronal space indicate edema 32.

Neuronal degeneration in experimental rats can be detected as early as 10 min after injury in the neocortex, thalamus, and hippocampus ipsilateral to the injury site 33. Degenerated neurons increased significantly 24 h following trauma and were localized primarily in the injured cortex 34. It was found that CNS injury triggers the release of numerous proinflammatory cytokines and other molecules that eventually result in apoptosis and necrosis of neurons, oligodendrocytes, and endothelial cells 35,36. The major pathophysiologic alterations resulting from a concussive brain injury include sudden neuronal depolarization, release of excitatory neurotransmitters, ionic changes, impaired axonal function, altered cerebral blood flow, and glucose metabolism. These alterations can be correlated with periods of postconcussion vulnerability and with neurobehavioral abnormalities 37.

The significant decrease in astrocytes indicated by the significant reduction in the GFAP intensity in this study was in accordance with the results of Fawcett and colleagues 28,29. They reported that gliosis, which is the process of increased number of neuroglial cells, comprises successive events that occur over several days. Microgliosis starts within hours of CNS injury, followed by oligodendrocyte proliferation and recruitment 3–5 days later, and then finally astrocytosis, which aids glial scar formation. Numerous apoptotic astrocytes and oligodendrocytes were added to the damaged cortex 1 week after trauma. As these processes develop over several hours, they can potentially be influenced by an early intervention 10,38.

The present study found a statistically significant increase in degenerated neurons and a decrease in the intensity of the SYN reaction in the concussion group than in the control group. This reduction in the SYN reaction was correlated with the degree of degeneration or neuronal loss of pyramidal cells in the third layer of the frontal cortex. A selective bilateral neuronal cell loss in the mice cerebral cortex and hippocampus but not in other brain regions was detected 24 h after trauma using Nissl staining (cresyl violet). The characteristics of neuronal cell loss in the cortex suggested that this pathology was related in part to the head impact dynamics 39.

In the recovery group of the current work, some neurons still showed degeneration and the frontal cortex showed hypercellularity, improvement in edema, and cellular infiltration, together with increased GFAP immunoreactivity. The increase in astrocytes detected could be because of an angiogenic response of the CNS to GFAP. The extensive increase in neuroglial cells causes considerable distortion of surrounding neuronal tissue. Astrogliosis is characterized by hypertrophy and hyperplasia of astrocytes, which markedly upregulate the expression of GFAP, an intermediate filament protein expressed in astrocytic cell bodies and processes 40,41. In contrast, a milder form and more organized increase in neuroglial cells were detected in the EPO-treated group. Mild or moderate reactive astrogliosis is generally associated with mild nonpenetrating and noncontusive trauma. If the triggering mechanism is able to resolve, then mild or moderate reactive astrogliosis shows the potential for resolution; however, its physiological consequences are not well understood 42.

In the EPO-treated group in the present study, the number of degenerated neurons was significantly decreased. The frontal cortex regained its normal organization and appearance and the pyramidal cells restored their normal structure. The mechanism by which EPO promotes neuronal survival after hypoxia and other metabolic insults is still unknown. As apoptosis and necrosis have been proposed as mechanisms of cellular death, either of them could be the target of actions of EPO. EPO not only prevents neuronal apoptosis but also has neurotrophic activity on primary motor neurons 43.

The present study found a significant decrease in GFAP in the treated group compared with the recovery group. Normally, the neuronal function in the brain is mediated in part by complex interactions among different cell types including astrocytes. Astrocytes outnumber brain neurons by over 5:1 and constitute 25–50% of brain volume. They function in energetic, antioxidant, and signaling processes required for normal functioning of the CNS 42. Astrocytes play a major role in neuroprotection and glutathione production, which is the most abundant intracellular nonprotein thiol and antioxidant in the body, especially the brain 44. EPO has recently been found to promote the development and survival of neurons and astrocytes 45.

Data from different models of neuronal injury suggest that EPO mediates its effects by a combination of mechanisms: (a) promotion of cell survival signaling cascades, thus reducing apoptosis 43, (b) decrease of intracellular calcium, (c) stimulation of nitric oxide production 46, (d) antioxidative 47, (e) anti-inflammatory 43, and (f) angiogenic actions 48.

The protective effects were detected when EPO was administered 6 h after the injury similar to previous studies 8. Some investigators described duration of the protective effects for at least 3 days 43. However, others suggested that a single dose of recombinant EPO was associated with the same excellent outcome as three EPO doses administered on successive days 8.

In experimental subarachnoidal hemorrhage, EPO was found to normalize cerebral blood flow 49. EPO both actively reduces the cerebral ischemia after hemorrhage by maintaining tissue perfusion and directly provides neuroprotection for metabolically stressed neurons 8. A marked reduction in the subpial space was detected in the EPO-treated group as compared with the nontreated group. Previous findings indicate that early post-traumatic administration of EPO reduces brain edema development until at least 6 h after injury and improves neurologic recovery. EPO can thus be considered as a potential agent in the treatment of TBI-induced diffuse edema. This antiedematous effect of EPO was possibly mediated through an early inhibition of extracellular-regulated kinase-1/-2 phosphorylation 50.

Significant restoration of SYN after trauma was observed in the EPO-treated group compared with the recovery group. EPO was found to decrease the excitatory neurotransmitter release probably following trauma and may in this way protect the synapses from toxic levels of glutamate 51. Moreover, EPO enhanced endothelial proliferation and the level of SYN expression in the brain of an Alzheimer disease rat model 52.

It could thus be concluded that early systemic administration of EPO, within days after concussion, produced a major neurological benefit. Thus, EPO could be a promising agent in protecting the brain tissue against the deleterious effect of repeated brain concussions.

T2-11
Table:
No title available.

Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

References

1. Badre D, D’Esposito M.Is the rostro-caudal axis of the frontal lobe hierarchical?Nat Rev Neurosci2009;10:659–669.
2. Pearce JMS.Observations on concussion: a review.Eur Neurol2008;59:113–119.
3. Langlois JA, Rutland-Brown W, Wald MM.The epidemiology and impact of traumatic brain injury: a brief overview.J Head Trauma Rehabil2006;21:375–378.
4. Bigler ED.Neuropsychology and clinical neuroscience of persistent post-concussive syndrome.J Int Neuropsychol Soc2008;14:1–22.
5. Chen Z, Leung LY, Mountney A, Liao Z, Yang W, Lu X-CM, et al..A novel animal model of closed-head concussive-induced mild traumatic brain injury: development, implementation, and characterization.J Neurotrauma2012;29:268–280.
6. Hylin MJ, Orsi SA, Rozas NS, Hill JL, Zhao J, Redell JB, et al..Repeated mild closed head injury impairs short-term visuospatial memory and complex learning.J Neurotrauma2013;30:716–726.
7. Xiong Y, Mahmood A, Chopp M.Emerging treatments for traumatic brain injury.Expert Opin Emerg Drugs2009;14:67–84.
8. Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, et al..Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma.Proc Natl Acad Sci USA2002;99:9450–9455.
9. Jelkmann W.Erythropoietin after a century of research: younger than ever.Eur J Haematol2007;78:183–205.
10. Haroon ZA, Amin K, Jiang X, Arcasoy MO.A novel role for erythropoietin during fibrin-induced wound-healing response.Am J Pathol2003;163:993–1000.
11. Strunk T, Härtel C, Schultz C.Does erythropoietin protect the preterm brain?Arch Dis Child Fetal Neonatal Ed2004;89:F364–F366.
12. Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG.Responses to cortical injury: I. Methodology and local effects of contusions in the rat.Brain Res1981;211:67–77.
13. Huh JW, Widing AG, Raghupathi R.Midline brain injury in the immature rat induces sustained cognitive deficits, bihemispheric axonal injury and neurodegeneration.Exp Neurol2008;213:84–92.
14. Metz GAS, Curt A, Van De Meent H, Klusman I, Schwab ME, Dietz V.Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury.J Neurotrauma2000;17:1–17.
15. Von Gertten C, Morales AF, Holmin S, Mathiesen T, Nordqvist A-CS.Genomic responses in rat cerebral cortex after traumatic brain injury.BMC Neurosci2005;6:69.
16. Bouzat P, Francony G, Thomas S, Valable S, Mauconduit F, Fevre M-C, et al..Reduced brain edema and functional deficits after treatment of diffuse traumatic brain injury by carbamylated erythropoietin derivative.Crit Care Med2011;39:2099–2105.
17. Bancroft JD, Gamble M.Theory and practice of histological techniques2008:6th ed..London:Churchill Livingstone;121–433.
18. Martin PM, O’Callaghan JP.A direct comparison of GFAP immunocytochemistry and GFAP concentration in various regions of ethanol-fixed rat and mouse brain.J Neurosci Methods1995;58:181–192.
19. Yao I, Iida J, Nishimura W, Hata Y.Synaptic and nuclear localization of brain-enriched guanylate kinase-associated protein.J Neurosci2002;22:5354–5364.
20. Graham L, Orenstein JM.Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research.Nat Protoc2007;2:2439–2450.
21. Fazeli SA, Gharravi AM, Jahanshahi M, Ghafari S, Behnampour N, Golalipour MJ.Resistance of CA1 pyramidal cells to STZ-induced diabetes in young rats.Int J Morphol2009;27:997–1001.
22. Gao Y, Bezchlibnyk YB, Sun X, Wang J-F, McEwen BS, Young LT.Effects of restraint stress on the expression of proteins involved in synaptic vesicle exocytosis in the hippocampus.Neuroscience2006;141:1139–1148.
23. Huh JW, Raghupathi R.Chronic cognitive deficits and long-term histopathological alterations following contusive brain injury in the immature rat.J Neurotrauma2007;24:1460–1474.
24. Ciurea AV, Gorgan MR, Tascu A, Sandu AM, Rizea RE.Traumatic brain injury in infants and toddlers, 0–3 years old.J Med Life2011;4:234–243.
25. Yang Q, Kock ND.Intestinal adaptation following massive ileocecal resection in 20-day-old weanling rats.J Pediatr Gastroenterol Nutr2010;50:16–21.
26. Saatman KE, Feeko KJ, Pape RL, Raghupathi R.Differential behavioral and histopathological responses to graded cortical impact injury in mice.J Neurotrauma2006;23:1241–1253.
27. Adelson PD, Dixon CE, Kochanek PM.Long-term dysfunction following diffuse traumatic brain injury in the immature rat.J Neurotrauma2000;17:273–282.
28. Fawcett JW, Asher RA.The glial scar and central nervous system repair.Brain Res Bull1999;49:377–391.
29. Streit WJ, Walter SA, Pennell NA.Reactive microgliosis.Prog Neurobiol1999;57:563–581.
30. Graeber MB, Blakemore WF, Kreutzberg GWGraham DI, Lantos PL.Cellular pathology of the central nervous system.Greenfield’s neuropathology2002:7th ed..London:Arnold;123–191.
31. Kadar T, Shapira S, Cohen G, Sahar R, Alkalay D, Raveh L.Sarin-induced neuropathology in rats.Hum Exp Toxicol1995;14:252–259.
32. Zhu H-L, Luo W-Q, Wang H.Iptakalim protects against hypoxic brain injury through multiple pathways associated with ATP-sensitive potassium channels.Neuroscience2008;157:884–894.
33. Hicks R, Soares H, Smith D, McIntosh T.Temporal and spatial characterization of neuronal injury following lateral fluid-percussion brain injury in the rat.Acta Neuropathol1996;91:236–246.
34. Conti AC, Raghupathi R, Trojanowski JQ, McIntosh TK.Experimental brain injury induces regionally distinct apoptosis during the acute and delayed post-traumatic period.J Neurosci1998;18:5663–5672.
35. Dusart I, Schwab ME.Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord.Eur J Neurosci1994;6:712–724.
36. Sekhon LHS, Fehlings MG.Epidemiology, demographics, and pathophysiology of acute spinal cord injury.Spine2001;2624 SupplS2–S12.
37. Giza CC, Hovda DA.The neurometabolic cascade of concussion.J Athletic Training2001;36:228–235.
38. Ruiyun P, Dewen W, Yabing G.Studies on pathologic changes of cerebral concussion and apoptosis of nervous cells in Wistar rats.Jie Fang Jun Yi Xue Za Zhi2002;27:991–992.
39. Tang Y-P, Noda Y, Hasegawa T, Nabeshima T.A concussive-like brain injury model in mice (II): selective neuronal loss in the cortex and hippocampus.J Neurotrauma1997;14:863–874.
40. Kimelberg HK, Jalonen T, Walz WMurphy S.Regulation of the brain microenvironment: transmitters and ions.Astrocytes: pharmacology and function1993.San Diego, CA:Academic Press;193–222.
41. Eng LF, Ghirnikar RS.GFAP and astrogliosis.Brain Pathol1994;4:229–237.
42. Sofroniew MV, Vinters HV.Astrocytes: biology and pathology.Acta Neuropathol2010;119:7–35.
43. Sirén A-L, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, et al..Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress.Proc Natl Acad Sci USA2001;98:4044–4049.
44. Narasimhan M, Rathinam M, Patel D, Henderson G, Mahimainathan L.Astrocytes prevent ethanol induced apoptosis of Nrf2 depleted neurons by maintaining GSH homeostasis.Open J Apoptosis2012;1:9–18.
45. Mohyeldin A, Dalgard CL, Lu H, Mcfate T, Tait AS, Patel VC, et al..Survival and invasiveness of astrocytomas promoted by erythropoietin.J Neurosurg2007;106:338–350.
46. Genc S, Kuralay F, Genc K, Akhisaroglu M, Fadiloglu S, Yorukoglu K, et al..Erythropoietin exerts neuroprotection in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated C57/BL mice via increasing nitric oxide production.Neurosci Lett2001;298:139–141.
47. Sela S, Shurtz-Swirski R, Sharon R, Manaster J, Chezar J, Shkolnik G, et al..The polymorphonuclear leukocyte – a new target for erythropoietin.Nephron2001;88:205–210.
48. Catania MA, Marciano MC, Parisi A, Sturiale A, Buemi M, Grasso G, et al..Erythropoietin prevents cognition impairment induced by transient brain ischemia in gerbils.Eur J Pharmacol2002;437:147–150.
49. Springborg JB, Ma X, Rochat P, Knudsen GM, Amtorp O, Paulson OB, et al..A single subcutaneous bolus of erythropoietin normalizes cerebral blood flow autoregulation after subarachnoid haemorrhage in rats.Br J Pharmacol2002;135:823–829.
50. Brown R, Thompson HJ, Imran SA, Ur E, Wilkinson M.Traumatic brain injury induces adipokine gene expression in rat brain.Neurosci Lett2008;432:73–78.
51. Kamal A, Al Shaibani T, Ramakers G.Erythropoietin decreases the excitatory neurotransmitter release probability and enhances synaptic plasticity in mice hippocampal slices.Brain Res2011;1410:33–37.
52. Lee S-T, Chu K, Park J-E, Jung K-H, Jeon D, Lim J-Y, et al..Erythropoietin improves memory function with reducing endothelial dysfunction and amyloid-beta burden in Alzheimer’s disease models.J Neurochem2012;120:115–124.
Keywords:

concussion; erythropoietin; glial fibrillar acid protein; histology; synaptophysin; young albino rats

© 2013 The Egyptian Journal of Histology