Traumatic brain injury (TBI) is a worldwide endemic that results in unacceptably high morbidity and mortality. Secondary injury processes following primary injury are composed of intricate interactions between assorted molecules that ultimately dictate the degree of longer-term neurological deficits. One comparatively unexplored molecule that may contribute to exacerbation of injury or enhancement of recovery is the posttranslationally modified polysialic acid form of neural cell adhesion molecule, PSA-NCAM. This molecule is a critical modulator of central nervous system plasticity and reorganization after injury. In this study, we used controlled cortical impact (CCI) to produce moderate or severe TBI in the mouse. Immunoblotting and immunohistochemical analysis were used to track the early (2, 24, and 48 hour) and late (1 and 3 week) time course and location of changes in the levels of PSA-NCAM after TBI. Variable and heterogeneous short- and long-term increases or decreases in expression were found. In general, alterations in PSA-NCAM levels were seen in the cerebral cortex immediately after injury, and these reductions persisted in brain regions distal to the primary injury site, especially after severe injury. This information provides a starting point to dissect the role of PSA-NCAM in TBI-related pathology and recovery.
Traumatic brain injury (TBI) is a leading cause of death and disability throughout the world. In the United States alone, the Centers for Disease Control (CDC) report that of the nearly 1.7 million people who suffer TBI each year, over 50,000 will die [
Studies aimed at identifying molecular targets that may improve survivability and decrease disability following TBI are manifold. However, in spite of decades of research and promising results in preclinical animal studies, no single effective therapy has successfully and consistently transferred to human clinical trials [
One molecule that may serve as a harbinger of the complex balance between central nervous system (CNS) postinjury amelioration and exacerbation is neural cell adhesion molecule (NCAM). Described over 30 years ago, NCAM was the first cell adhesion molecule (CAM) identified [
Of paramount importance in maintaining the differentiation, migration, and anchoring function of NCAM during development and neurogenesis and cell survival functions in the face of insult during adulthood is the degree of expression of the posttranslationally glycosylated NCAM isoforms manifesting the large, negatively charged
Considering the essential contributions of PSA-NCAM to efficient CNS function, it may be presumed that, at least in part, alterations in expression levels and function are involved in the complex pathology that follows TBI. To date, the short- and long-term alterations in expression levels of PSA-NCAM following graded-controlled cortical impact (CCI) in the mouse have not been characterized. The current study examines the immediate (two, 24, and 48 hour) and long-term (one and three week) change in the expression levels of PSA-NCAM in eight distinct regions of the mouse brain following graded-CCI that results in either moderate or severe injury. An understanding of the time course and regional distribution of PSA-NCAM alterations following TBI may contribute to the knowledge base that is being assembled to identify novel therapeutic targets.
All experimental procedures used in this investigation were approved by the Uniformed Services University of the Health Sciences (USUHS) Institutional Animal Care and Use Committee (IACUC). Six- to nine-week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) weighing 20–29 g were housed in an animal colony at a constant temperature (23 ± 2°C) with a 12-hour light/dark cycle and food and water
Induction of mice was performed via spontaneous ventilation using 3% isoflurane in 100% oxygen (1.0 L per minute flow rate) for 3 minutes in a rodent volatile anesthesia box. After the application of protective ointment (Lacri-Lube) to the eyes, the head was shaved using electric clippers. Following hair removal, the head of the animal was placed in a standard stereotaxic frame and positioned using ear and incisor bars (Stoelting, Wood Dale, IL) and the skin was prepped with betadine ointment. Following skin preparation, 0.1 mL of 0.025% bupivacaine was injected subcutaneously into the planned incision site. Rectal temperature was maintained at 37°C with an isothermal heating pad and feedback controller (Stoelting, Wood Dale, IL). Anesthesia was maintained with 1.5–2% isoflurane. Unilateral CCI to the left cerebral hemisphere was performed using a modified technique previously described [
Twenty-four hours following graded-CCI, four sham, eight moderate, and four severe injury mice were administered deep anesthesia (60 mg/kg ketamine with 60 mg/kg xylazine, IP). The animals were perfused intracardially with heparinized (1,000 u/L) 0.9% sodium chloride followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Following decapitation, two sham, six moderate, and two severe injury brains were harvested for whole brain and coronal video microscopy (Dazor Speck Finder, St. Louis, MO, USA). Additionally, one moderate and one severe injury animal was sacrificed at three weeks. Whole brain and 1 mm coronal sections through the epicenter of the impact site were imaged to assess the extent of hemorrhage and gross tissue disruption in the brain. The remaining brains were postfixed in the skull overnight, and then removed from the skull and sequentially cryoprotected in 20 and 30% sucrose in phosphate buffer saline (PBS) until the brains sank. A frozen sliding microtome was used to acquire 30-
Western blot analysis was performed as previously described [
At the designated harvest times, additional mice were euthanized (60 mg/kg ketamine with 60 mg/kg xylazine, IP) and brains were processed for immunohistochemical analysis using a modified technique previously described [
For PSA-NCAM immunohistochemical evaluation, with diaminobenzidine as the chromogen, a modified technique previously described was utilized [
Quantitative western blot measurements of PSA-NCAM were normalized by dividing optical density values by corresponding measures of
Examination of tissue samples obtained 24 hours after injury revealed a progressively larger hemorrhagic lesion of the brain in the graded-CCI animals that correlated well with the depth of impact. Sham animals demonstrated no obvious injury (Figures
Graded injury severity. Whole brain and coronal sections displaying injury severity at 24 hours in sham (a), (b), moderate (c), (d), and severe (e), (f) injury groups. Coronal H&E sections revealed no apparent hemorrhage in the underlying hippocampus in the moderate injury animals 24 hours following injury (g). Severe injury revealed extensive hippocampal and thalamic injury at the 24 hour collection point (h). Lesions on the cerebral surface were evident three weeks following severe injury (i). Moderate injury whole brain samples displayed no apparent surface defect three weeks following injury (j). Coronal sections from brain samples obtained 3 weeks after severe CCI revealed graded substantial cortical and hippocampal damage (k) while moderate CCI resulted in loss of cortical tissue and deformity and expansion of hippocampal neuropil (l). Arrows denote region of interest. Coronal sections approximate bregma −1.70. Scale bar equals 1 mm.
Western blot analysis of acute PSA-NCAM level alterations following moderate TBI revealed heterogeneity as a function of brain region (Figure
Quantitative analysis of PSA-NCAM levels following graded-CCI. Short- and long-term changes in expression levels of PSA-NCAM in the eight brain regions evaluated using western blotting. Graphs depict mean PSA-NCAM density differences ± SEM of injury groups compared to the sham group (adjusted to mean = 1). Star color indicates statistical significance of difference between injury and sham values via one-way ANOVA and
PSA-NCAM levels were evaluated one and three weeks after CCI (Figure
Immunoblot results for PSA-NCAM one week after CCI. One week western blot results of the eight brain regions examined following sham, moderate, or severe injury illustrate a heterogeneous response to injury. PSA-NCAM blotting is noted to span from approximately 120–250 KDa. The appearance of bands of different intensity is the result of variable degrees of polysialylation of NCAM 180, 140, and 120 (approximate weight noted on right side of image). The results represented in this figure were used to construct the one week data in Figure
Qualitative evaluation of tissue slices was undertaken to further examine PSA-NCAM changes seen with western blotting. The approach was to carefully survey all regions of brain sections that were obtained at the indicated time points after injury that we believed would produce large enough alterations in expression as to be detected using microscopy. In spite of attempts to process tissue sections identically, variability within groups was observed, so that qualitative findings intermittently supported the trends revealed with immunoblotting. For example, the two hour samples from immunoblot analysis indicated that PSA-NCAM expression decreased in the right cortex of both injury groups at this time point, but a noticeable difference between groups was not reliably observed using immunohistochemical evaluation (data not shown). Likewise, PSA-NCAM expression in the medial prefrontal cortex did not appear to be different between groups. These observations are partially anticipated as the general PSA-NCAM expression pattern in most of the cortex does not show a distinct structural distribution (with exceptions; see below for temporal lobe PSA-NCAM staining) and appears as faint labeling of neuropil or immunonegative [
Comparison of PSA-NCAM levels in right dentate gyrus of sham and severe injury animals 48 hours following surgery. PSA-NCAM staining of subgranular zone cells in the contralateral hippocampus reveals more intense expression in the soma (arrows) and processes (arrow heads) of sham (a) versus severe (b) injury animals. Images are 40(x) magnification; scale bar = 100
Immunohistochemical evidence of PSA-NCAM expression alterations in the left cortex one week following injury. (a) Sham animals demonstrate subtle, uniform PSA-NCAM staining in the left cortex one week following craniectomy. (b) PSA-NCAM expression in the ipsilateral cortex of moderate injury animals is significantly increased with particularly dark staining of cells that display the appearance of reactive astrocytes (arrows). Images are 20(x) magnification; scale bar = 300
PSA-NCAM staining in left temporal lobe one-week following graded-CCI. Diaminobenzidine staining for PSA-NCAM in sham (a), moderate (b), and severe (c) injury animals one week after CCI demonstrates decreased staining of the soma (large arrows) and apical dendrites (small arrows) of neurons in the piriform cortex. Insert denotes approximate region of interest at approximately bregma −1.70 mm. Images are 20(x) magnification; scale bar = 300
Originally thought to be only expressed in the CNS during embryonic development [
The qualitative and quantitative distribution of PSA-NCAM in the uninjured adult CNS has been examined and characterized in previous studies [
Following injury in the parietal cortex, levels exhibited fluctuations that included an initial increase in PSA-NCAM levels in the region of primary impact in the left parietal cortex in moderately-injured animals, while levels were depressed when the injury was severe. Correspondingly, in the contralateral hemisphere, PSA-NCAM reductions in the corresponding parietal cortex remained for a longer time after moderate injury and were persistently depressed after severe CCI. Perhaps the final outcome following CCI is telling. At three weeks after CCI, levels of PSA-NCAM were elevated in the left parietal lobe that sustained primary injury, while levels were reduced in the right parietal cortex and the diencephalon. In the left temporal lobe, levels were not reduced during the initial two days after injury, but then were depressed by the first and third week post-injury. However, in the contralateral temporal lobe, PSA-NCAM levels were unaffected by the injury. These data may belie the overall direct versus indirect connections of the injured parietal cortex with proximal and more remote structures. Specifically, by three weeks after injury there was a “local” increase in PSA-NCAM levels as a result of reorganization at the primary site of injury, while regions with more direct and robust connections to the injured site (the thalamus, the contralateral parietal region, and the ipsilateral temporal lobe) exhibited diminished levels. Conversely, the more distal, contralateral temporal lobe appears to have been unaffected in terms of PSA-NCAM levels. Although we were unable to fully corroborate the PSA-NCAM changes seen in the hippocampus via western blotting efforts with the results obtained using IHC, these alterations too may have implications in long term recovery. For example, the immunoblot data indicated an increase in PSA-NCAM levels at one week in either the ipsilateral or contralateral hippocampi following severe or moderate TBI, respectively. Even though variability in the intensity of staining prevented us from isolating the location and degree of these changes, these alterations may indicate the response of a group of immature neurons in the subgranular zone reacting to injury [
While the present results are one of the first to examine PSA-NCAM alterations after controlled cortical impact, the role of PSA-NCAM in prosurvival pathways initiated during CNS excitotoxicity insult has been investigated [
PSA-NCAM’s involvement in cell signaling is both far reaching and essential for proper nervous system function. Figure
NCAM and PSA-NCAM signaling pathways. Currently accepted signaling pathways believed to be involved in neurite outgrowth; the most studied and understood result of NCAM activation. NCAM-180 and- 140 are represented by five Ig like domains and two fibronectin III domains extracellularly and an intracellular segment of varying length. NCAM-120 is attached to the membrane via a GPI anchor. Color designations: tyrosine kinases = red; other protein kinases = blue; nonproteins = silver. Abbreviations: AA, arachidonic acid; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; CaMK, Ca2+-calmodulin-dependent kinase; cGMP, cyclic guanosine monophosphate; CKII, casein kinase II; DAG, diacylglycerol; ERK, extracellular regulated kinase; FAK, focal adhesion kinase; FGFR, fibroblast growth factor receptor; Frs2, FGFR substrate; GAP-43, growth-associated protein 43; NO, nitric oxide; NOS, NO synthase; NSCC, nonspecific cation channel; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PLC, phospholipase C; RPTP, receptor protein tyrosine phosphatase; VDCC, voltage-dependent Ca2+-channel. Broken lines indicate putative intracellular interactions. Illustration adapted with permission from [
Do the alterations in PSA-NCAM expression following TBI present as possible targets of therapy? The ability of PSA to modulate cell interactions in order to promote plasticity, precursor cell migration, and axonal defasciculation and targeting, raises the possibility of using PSA gain-of-function to augment the regenerative response of the brain to injury. Our study reveals several potential therapeutic treatment windows for PSA therapy to promote function of endogenous recovery mechanisms while combating the postinjury inhibitory environment in the brain. For example, PSA-NCAM expression was significantly decreased three weeks following injury in multiple brain regions including the cerebral and temporal cortices, hippocampus, diencephalon, and cerebellum following moderate or severe injury. In some cases, this decrease followed an earlier period of increased expression of PSA-NCAM (Figure
Traumatic brain injury is a devastating and life altering event that is sharply increasing in incidence throughout the world, particularly among military members, as well as in civilian populations in developing countries where there is a recent increase in motor vehicle use [
In this study we examined changes in PSA-NCAM expression following graded-CCI in the mouse. Severe and moderate injury produced immediate as well as long-term alterations in PSA-NCAM expression both proximal and distal to the impact site. Alterations in these species of adhesion molecule have been shown to result in acute and long-lasting alterations in neuron migration, neurite formation and axon fasciculation, synapse development and function, memory function, and emotional status. The significant expression level changes seen in our study may contribute to dysfunction and/or healing following injury.
This work was supported by TriService Nursing Research Program Grant N10-P09 to C. S. Budinich and US Army Medical Research and Materiel Command grant W81XWH-09-2-0147 and a USUHS Concept Award RO704B to J. T. MCabe. The experiments were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, ILAR, National Research Council, DHEW Publication no. (NIH) 73-23. The opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the US Army, Department of Defense, the US government, or the Uniformed Services University of the Health Sciences. The use of trade names does not constitute an official endorsement or approval of the use of such reagents or commercial hardware or software. This paper may not be cited for purposes of advertisement. The investigators appreciated utilization of the preclinical core facilities of the Center for Neuroscience and Regenerative Medicine, USUHS. The authors thank Mr. Eric Prager for assistance with the construction of Figure