Comparison of Ionized Calcium-binding Adapter Molecule 1 Immunoreactivity of the Hippocampal Dentate Gyrus and CA1 Region in Adult and Aged Dogs

Similarities between age-related changes in the canine and human brain have resulted in the general acceptance of the canine brain as a model of human brain aging. The hippocampus is essentially required for intact cognitive ability and appears to be particularly vulnerable to the aging process. We observed changes in ionized calcium-binding adapter molecule 1 (Iba-1, a microglial marker) immunoreactivity and protein levels in the hippocampal dentate gyrus and CA1 region of adult (2-3 years) and aged (10-12 years) dogs. We also observed the interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, protein levels in these groups. In the dentate gyrus and CA1 region of the adult dog, Iba-1 immunoreactive microglia were well distributed and their processes were highly ramified. However, in the aged dog, the processes of Iba-1 immunoreactive microglia were hypertrophied in the dentate gyrus. Moreover, Iba-1 protein level in the dentate gyrus in the aged dog was higher than in the adult dog. IFN-gamma expression was increased in the dentate gyrus homogenates of aged dogs than adult dogs. In addition, we found that some neurons were positive to Fluoro-Jade B (a marker for neuronal degeneration) in the dentate polymorphic layer, but not in the hippocampal CA1 region in the aged dog. These results suggest that Iba-1 immunoreactive microglia are hypertrophied in the dentate gyrus in the aged dog.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Comparison of GAD65 and 67 Immunoreactivity in the Lumbar Spinal Cord Between Young Adult and Aged Dogs

We investigated distribution and age-related changes in two isoforms of GABA synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and 67, in the lumbar levels (L(5)-L(6)) of the dog spinal cord. Male German shepherds were used at 1-2 years (young adult dogs) and 10-12 years (aged dogs) of age. GAD65 immunoreaction was observed in neuropil, not in cell bodies, in all laminae of the adult lumbar spinal cord: Many punctate GAD65-immunoreactive structures were shown in all laminae. The density of GAD65 immunoreactive structures was highest in laminae I-III, and lowest in lamina VII. In the aged dog, the distribution pattern of GAD65 immunoreactivity was similar to that in the adult dog; however the density of GAD65-immunoreactive structures and its protein levels were significantly increased in the aged lumbar spinal cord. GAD67 immunoreaction in the adult dog was also distributed in all laminae of the lumbar spinal cord like GAD65; however, we found that small GAD67-immunoreactive cell bodies were observed in laminae II, III and VIII. In the aged dogs, GAD67 immunoreactivity and its protein levels were also increased compared to those in the adult group. In conclusion, our results indicate that the distribution of GAD65-immunoreactive structures is different from GAD67-immunoreactive structures and that their immunoreactivity in the aged dogs is much higher than the adult dogs.     

Comparison of Glucocorticoid Receptor and Ionized Calcium-Binding Adapter Molecule 1 Immunoreactivity in the Adult and Aged Gerbil Hippocampus Following Repeated Restraint Stress

Stress leads to changes in homeostasis and internal balance of the body and is known to be one of important factors in the development of several diseases. In the present study, we investigated changes in glucocorticoid receptor (GR) and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity in the adult and aged gerbil hippocampus after chronic restraint stress. Serum corticosterone level was much higher in both the stress-groups than the control groups. No neuronal death was found in all hippocampal subregions of the adult and aged gerbil after chronic restraint stress. GR immunoreactivity was decreased in both the adult and aged groups after repeated restraint stress; however, GR immunoreactivity in the adult-stress-group was decreased much more than that in the aged-stress-group. Iba-1 immunoreactive microglia were hypertrophied and activated in the adult group after repeated restraint stress; in the aged-stress-group, there was no any significant change in Iba-1 immunoreactive microglia. In brief, level of GR, not Iba-1, in the hippocampus was much decreased in the adult gerbil compared to the aged gerbil following chronic restraint stress.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

Comparison of Alpha-Synuclein Immunoreactivity in the Hippocampus Between the Adult and Aged Beagle Dogs

Alpha-synuclein (α-syn), as a neuroprotein, is expressed in neural tissue, and it is related to a synaptic transmission and neuronal plasticity. In this study, we compared the distribution and immunoreactivity of α-syn and related gliosis in hippocampus between young adult (2–3 years) and aged (10–12 years) beagle dogs. In both groups, α-syn immunoreactivity was detected in neuropil of all the hippocampal sub-regions, but not in neuronal somata. In the aged hippocampus, α-syn immunoreactivity was apparently increased in mossy fibers compared to that in the adult dog. In addition, α-syn protein level was markedly increased in the aged hippocampus. On the other hand, GFAP and Iba-1 immunoreactivity in astrocytes and microglia, respectively, were increased in all the hippocampal sub-regions of the aged group compared to that in the adult group: especially, their immunoreactivity was apparently increased around mossy fibers. In addition, in this study, we could not find any expression of α-syn in astrocytes and microglia. These results indicate that α-syn immunoreactivity apparently increases in the aged hippocampus and that GFAP and Iba-1 immunoreactivity are also apparently increased at the regions with increased α-syn immunoreactivity. This increase in α-syn expression might be a feature of normal aging.     

Age-Related Changes in Microglia of the Rat Spinal Cord

Journal of Evolutionary Biochemistry and Physiology - The aim of this study was to evaluate age-related changes in microglial cells in various regions of the cervical spinal cord in young (4...     

Differences in Doublecortin Immunoreactivity and Protein Levels in the Hippocampal Dentate Gyrus Between Adult and Aged Dogs

Doublecortin (DCX), a microtubule-associated protein, specifically expresses in neuronal precursors. This protein has been used as a marker for neuronal precursors and neurogenesis. In the present study, we observed differences in DCX immunoreactivity and its protein levels in the hippocampal dentate gyrus between adult and aged dogs. In the adult dog, DCX immunoreactive cells with well-stained processes were detected in the subgranular zone of the dentate gyrus. Numbers of DCX immunoreactive cells in the dentate gyrus of the aged dog were significantly decreased compared to those in the adult dog. DCX immunoreactive cells in both adult and aged dog did not show NeuN (a marker for mature neurons) immunoreactivity. NeuN immunoreactivity in the aged dog was poor compared to that in the adult dog. DCX protein level in the aged dentate gyrus was decreased by 80% compared to that in the adult dog. These results suggest that the reduction of DCX in the aged hippocampal dentate gyrus may be involved in some neural deficits related to the hippocampus.     

Cervical Spinal Cord Atrophy Profile in Adult SMN1-Linked SMA

PurposeThe mechanisms underlying the topography of motor deficits in spinal muscular atrophy (SMA) remain unknown. We investigated the profile of spinal cord atrophy (SCA) in SMN1-linked SMA, and its correlation with the topography of muscle weakness.ResultsCSA measurements revealed a significant cord atrophy gradient mainly located between C3 and C6 vertebral levels with a SCA rate ranging from 5.4% to 23% in SMA patients compared to controls. RD was significantly lower in SMA patients compared to controls in the anterior-posterior direction with a maximum along C4 and C5 vertebral levels (p-values < 10⁻⁵). There were no correlations between atrophy measurements, strength and disability scores.ConclusionsSpinal cord atrophy in adult SMN1-linked SMA predominates in the segments innervating the proximal muscles. Additional factors such as neuromuscular junction or intrinsic skeletal muscle defects may play a role in more complex mechanisms underlying weakness in these patients.     

Efficacy of a Metalloproteinase Inhibitor in Spinal Cord Injured Dogs

Matrix metalloproteinase-9 is elevated within the acutely injured murine spinal cord and blockade of this early proteolytic activity with GM6001, a broad-spectrum matrix metalloproteinase inhibitor, results in improved recovery after spinal cord injury. As matrix metalloproteinase-9 is likewise acutely elevated in dogs with naturally occurring spinal cord injuries, we evaluated efficacy of GM6001 solubilized in dimethyl sulfoxide in this second species. Safety and pharmacokinetic studies were conducted in naïve dogs. After confirming safety, subsequent pharmacokinetic analyses demonstrated that a 100 mg/kg subcutaneous dose of GM6001 resulted in plasma concentrations that peaked shortly after administration and were sustained for at least 4 days at levels that produced robust in vitro inhibition of matrix metalloproteinase-9. A randomized, blinded, placebo-controlled study was then conducted to assess efficacy of GM6001 given within 48 hours of spinal cord injury. Dogs were enrolled in 3 groups: GM6001 dissolved in dimethyl sulfoxide (n = 35), dimethyl sulfoxide (n = 37), or saline (n = 41). Matrix metalloproteinase activity was increased in the serum of injured dogs and GM6001 reduced this serum protease activity compared to the other two groups. To assess recovery, dogs were a priori stratified into a severely injured group and a mild-to-moderate injured group, using a Modified Frankel Scale. The Texas Spinal Cord Injury Score was then used to assess long-term motor/sensory function. In dogs with severe spinal cord injuries, those treated with saline had a mean motor score of 2 (95% CI 0–4.0) that was significantly (P<0.05; generalized linear model) less than the estimated mean motor score for dogs receiving dimethyl sulfoxide (mean, 5; 95% CI 2.0–8.0) or GM6001 (mean, 5; 95% CI 2.0–8.0). As there was no independent effect of GM6001, we attribute improved neurological outcomes to dimethyl sulfoxide, a pleotropic agent that may target diverse secondary pathogenic events that emerge in the acutely injured cord.     

Glial Cell Composition and Ultrastructure of the Caudal Spinal Cord of Young and Adult Tuataras,Sphenodon punctatus

Research on the caudal spinal cord (SC) of three young and three adult tuataras (Sphenodon punctatus) has revealed that most of the glial cells were well differentiated as astrocytes and oligodendrocytes. The former type was not completely differentiated 3 months post-hatching, but was the main glial cell type at the age of 1 yr and in adulthood. Smaller numbers of oligodendrocytes were found in the white matter of adult animals than fibrous astrocytes. It was concluded that during growth, there is a progressive decrease in the dark, basophilic and electron-dense glioblasts, most of which develop as astrocytes.     

Promotion of Spinal Cord Regeneration by Neural Stem Cell-Secreted Trimerized Cell Adhesion Molecule L1

The L1 cell adhesion molecule promotes neurite outgrowth and neuronal survival in homophilic and heterophilic interactions and enhances neurite outgrowth and neuronal survival homophilically, i.e. by self binding. We investigated whether exploitation of homophilic and possibly also heterophilic mechanisms of neural stem cells overexpressing the full-length transmembrane L1 and a secreted trimer engineered to express its extracellular domain would be more beneficial for functional recovery of the compression injured spinal cord of adult mice than stem cells overexpressing only full-length L1 or the parental, non-engineered cells. Here we report that stem cells expressing trimeric and full-length L1 are indeed more efficient in promoting locomotor recovery when compared to stem cells overexpressing only full-length L1 or the parental stem cells. The trimer expressing stem cells were also more efficient in reducing glial scar volume and expression of chondroitin sulfates and the chondroitin sulfate proteoglycan NG2. They were also more efficient in enhancing regrowth/sprouting and/or preservation of serotonergic axons, and remyelination and/or myelin sparing. Moreover, degeneration/dying back of corticospinal cord axons was prevented more by the trimer expressing stem cells. These results encourage the view that stem cells engineered to drive the beneficial functions of L1 via homophilic and heterophilic interactions are functionally optimized and may thus be of therapeutic value.     

Comparison of Glial Activation in the Hippocampal CA1 Region Between The Young and Adult Gerbils After Transient Cerebral Ischemia

It has been reported that young animals are less vulnerable to brain ischemia. In the present study, we compared gliosis in the hippocampal CA1 region of the young gerbil with those in the adult gerbil induced by 5?min of transient cerebral ischemia by immunohistochemistry and western blot for glial cells. We used male gerbils of postnatal month 1 (PM 1) as the young and PM 6 as the adult. Neuronal death in CA1 pyramidal neurons in the adult gerbil occurred at 4?days posti-schemia; the neuronal death in the young gerbil occurred at 7?days post-ischemia. The findings of glial changes in the young gerbil after ischemic damage were distinctively different from those in the adult gerbil. Glial fibrillary acidic protein-immunoreactive astrocytes, ionized calcium-binding adapter molecule (Iba-1), and isolectin B4-immunoreactive microglia in the ischemic CA1 region were activated much later in the young gerbil than in the adult gerbil. In brief, very less gliosis occurred in the hippocampal CA1 region of the young gerbil than in the adult gerbil after transient cerebral ischemia.     

Phosphorylation of Extracellular Signal-Regulated Kinases 1/2 Predominantly Enhanced in the Microglia of the Rat Spinal Cord Following Lipopolysaccharide Injection

The present study was initiated to investigate the role of extracellular signal-regulated kinases (ERK) 1/2 signaling pathway in the early response of spinal cord to systemic inflammation by using Western blotting and immunohistochemical techniques in a rat model intraperitoneally injected with 10 mg/kg of lipopolysaccharide (LPS). The results showed that there was a considerable amount of phosphorylated ERK 1/2 protein in the spinal cord of inflamed animals killed under pentobarbital anesthesia. The result of Western blotting showed that the phosphorylation level of ERK 1/2 in the spinal cord was increased at one hour; then 12 and 24 h after LPS injection the level decreased, while the total ERK 1/2 level seemed unchanged. The phosphorylated ERK 1/2 dominantly existed in the microglia cells of the gray matter of spinal cord, as demonstrated with double immunofluorescent staining 1 h after LPS injection. Collectively, the present results suggest that ERK signal pathway involve the cellular activation in the spinal cord following systemic inflammation, with ERK mainly in microglia. The increase of phosphorylation of ERK 1/2 in microglia of spinal cord after LPS injection implicates that ERK signaling pathway involves intracellular activity of microglia responding to the inflammation. Dan Zhou and Min Fei contributed equally to this work.     

Comparison of Neurogenesis in the Dentate Gyrus Between the Adult and Aged Gerbil Following Transient Global Cerebral Ischemia

In the present study, we compared differences in cell proliferation, neuroblast differentiation and neuronal maturation in the hippocampal dentate gyrus (DG) between the adult and aged gerbil induced by 5 min of transient global cerebral ischemia using Ki-67 and BrdU (markers for cell proliferation), doublecortin (DCX, a marker for neuroblast differentiation) and neuronal nuclei (NeuN, a marker for mature neuron). The number of Ki-67-immunoreactive (⁺) cells in the DG of both the groups peaked 7 days after ischemia/reperfusion (I/R). However, the number in the aged DG was 40.6 ± 1.8% of that in the adult DG. Thereafter, the number decreased with time. After ischemic damage, DCX immunoreactivity and its protein level in the adult and aged DG peaked at 10 and 15 days post-ischemia, respectively. However, DCX immunoreactivity and its protein levels in the aged DG were much lower than those in the adult. DCX immunoreactivity and its protein level in the aged DG were 11.1 ± 0.6% and 34.4 ± 2.1% of the adult DG, respectively. In addition, the number of Ki-67⁺ cells and DCX immunoreactivity in both groups were similar to those in the sham at 60 days postischemia. At 30 days post-ischemia, the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in the adult-group were much higher (281.2 ± 23.4% and 126.4 ± 7.4%, respectively) than the aged-group (35.6 ± 6.8% and 79.5 ± 6.1%, respectively). These results suggest that the ability of neurogenesis in the ischemic aged DG is much lower than that in the ischemic adult DG.     

Comparison of the Effects of Cuprizone on Demyelination in the Corpus Callosum and Hippocampal Progenitors in Young Adult and Aged Mice

Neurochemical Research - Cuprizone is commonly used to induce neuronal demyelination in mice. In the present study, we compared the cuprizone-induced demyelination in the corpus callosum and...     

Comparison of Trophic Factors Changes in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we investigated neuronal death/damage in the gerbil hippocampal CA1 region (CA1) and compared changes in some trophic factors, such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), in the CA1 between the adult and young gerbils after 5?min of transient cerebral ischemia. Most of pyramidal neurons (89?%) were damaged 4?days after ischemia?Creperfusion (I?CR) in the adult; however, in the young, about 59?% of pyramidal neurons were damaged 7?days after I?CR. The immunoreactivity and levels of BDNF and VEGF, not GDNF, in the CA1 of the normal young were lower than those in the normal adult. Four days after I?CR in the adult group, the immunoreactivity and levels of BDNF and VEGF were distinctively decreased, and the immunoreactivity and level of GDNF were increased. However, in the young group, all of their immunoreactivities and levels were much higher than those in the normal young group. From 7?days after I?CR, all the immunoreactivities and levels were apparently decreased compared to those of the normal adult and young. In brief, we confirmed our recent finding: more delayed and less neuronal death occurred in the young following I?CR, and we newly found that the immunoreactivities of trophic factors, such as BDNF, GDNF, and VEGF, in the stratum pyramidale of the CA1 in the young gerbil were much higher than those in the adult gerbil 4?days after transient cerebral ischemia.     

Immunoreactivities and Levels of Mineralocorticoid and Glucocorticoid Receptors in the Hippocampal CA1 Region and Dentate Gyrus of Adult and Aged Dogs

Corticosteroids are important factors in the maintenance of homeostasis in the brain. They are regulated via the interaction with two corticosteroid receptor systems—the mineralocorticoid (MR) and glucocorticoid receptor (GR). In the present study, we observed age-related changes in serum cortisol levels, and immunoreactivities and protein levels of MR and GR in the hippocampal CA1 region and dentate gyrus. The serum cortisol levels were significantly high (about twofold) in the aged group compared to that in the adult group. In the adult dog (2–3 years old), MR and GR immunoreactivity was detected in neurons in the pyramidal layer of the CA1 region, and in the granular and multiform layers of the dentate gyrus. In the aged dog (10–12 years old), MR immunoreactivity in the CA1 region was significantly decreased, especially, in the dentate multiform layer. In contrast, GR immunoreactivity in the aged dog was slightly decreased in the CA1 region and dentate gyrus. In the Western blot analysis, MR protein level in the aged dog was significantly lower compared to that of the adult dog; GR protein level in the aged dog was not significantly decreased. This result indicates that the reduction of MR immunoreactivity and protein level in the hippocampus of the aged dog may be associated with neural dysfunction in the aged hippocampus.     

Identification of Up-Regulated Genes After Complete Spinal Cord Transection in Adult Rats

Spinal cord injury (SCI) initiates a cascade of events and these responses to injury are likely to be mediated and reflected by changes in mRNA concentrations. As a step towards understanding the complex mechanisms underlying repair and regeneration after SCI, the gene expression pattern was examined 4.5 days after complete transection at T8-9 level of rat spinal cord. Improved subtractive hybridization was used to establish a subtracted cDNA library using cDNAs from normal rat spinal cord as driver and cDNAs from injured spinal cord as tester. By expressed sequence tag (EST) sequencing, we obtained 73 EST fragments from this library, representing 40 differentially expressed genes. Among them, 32 were known genes and 8 were novel genes. Functions of all annotated genes were scattered in almost every important field of cell life such as DNA repair, detoxification, mRNA quality control, cell cycle control, and signaling, which reflected the complexity of SCI and regeneration. Then we verified subtraction results with semiquantitative RT-PCR for eight genes. These analyses confirmed, to a large extent, that the subtraction results accurately reflected the molecular changes occurring at 4.5 days post-SCI. The current study identified a number of genes that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using improved subtractive hybridization may lead to a better understanding of molecular mechanisms responsible for repair and regeneration after SCI.