Astrocyte-Specific Overexpression of Insulin-Like Growth Factor-1 Protects Hippocampal Neurons and Reduces Behavioral Deficits following Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) survivors often suffer from long-lasting cognitive impairment that stems from hippocampal injury. Systemic administration of insulin-like growth factor-1 (IGF-1), a polypeptide growth factor known to play vital roles in neuronal survival, has been shown to attenuate posttraumatic cognitive and motor dysfunction. However, its neuroprotective effects in TBI have not been examined. To this end, moderate or severe contusion brain injury was induced in mice with conditional (postnatal) overexpression of IGF-1 using the controlled cortical impact (CCI) injury model. CCI brain injury produces robust reactive astrocytosis in regions of neuronal damage such as the hippocampus. We exploited this regional astrocytosis by linking expression of hIGF-1 to the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, effectively targeting IGF-1 delivery to vulnerable neurons. Following brain injury, IGF-1Tg mice exhibited a progressive increase in hippocampal IGF-1 levels which was coupled with enhanced hippocampal reactive astrocytosis and significantly greater GFAP levels relative to WT mice. IGF-1 overexpression stimulated Akt phosphorylation and reduced acute (1 and 3d) hippocampal neurodegeneration, culminating in greater neuron survival at 10d after CCI injury. Hippocampal neuroprotection achieved by IGF-1 overexpression was accompanied by improved motor and cognitive function in brain-injured mice. These data provide strong support for the therapeutic efficacy of increased brain levels of IGF-1 in the setting of TBI.     

Combined treatment with platelet-rich plasma and brain-derived neurotrophic factor-overexpressing bone marrow stromal cells supports axonal remyelination in a rat spinal cord hemi-section model

Background aimsCombining biologic matrices is becoming a better choice to advance stem cell-based therapies. Platelet-rich plasma (PRP) is a biologic product of concentrated platelets and has been used to promote regeneration of peripheral nerves after injury. We examined whether PRP could induce rat bone marrow stromal cells (BMSCs) differentiation in vitro and whether a combination of BMSCs, PRP and brain-derived neurotrophic factor (BDNF) could provide additive therapeutic benefits in vivo after spinal cord injury (SCI).MethodsBMSCs and BDNF-secreting BMSCs (BDNF-BMSCs) were cultured with PRP for 7 days and 21 days, respectively, and neurofilament (NF)-200, glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2) and ribosomal protein S6 kinase (p70S6K) gene levels were assessed. After T10 hemi-section in 102 rats, 15-μL scaffolds (PRP alone, BMSCs, PRP/BMSCs, BDNF-BMSCs or PRP/BDNF-BMSCs) were transplanted into the lesion area, and real-time polymerase chain reaction, Western blot, immunohistochemistry and ultrastructural studies were performed.ResultsThe messenger RNA expression of NF-200, GFAP, MAP2 and p70S6K was promoted in BMSCs and BDNF-BMSCs after culture with PRP in vitro. BDNF levels were significantly higher in the injured spinal cord after implantation of BDNF-BMSCs. In the PRP/BDNF-BMSCs group at 8 weeks postoperatively, more GFAP was observed, with less accumulation of astrocytes at the graft-host interface. Rats that received PRP and BDNF-BMSC implants showed enhanced hind limb locomotor performance at 8 weeks postoperatively compared with control animals, with more axonal remyelination.ConclusionsA combined treatment comprising PRP and BDNF-overexpressing BMSCs produced beneficial effects in rats with regard to functional recovery after SCI through enhancing migration of astrocytes into the transplants and axonal remyelination.     

Detection of sodium channel distribution in rat sciatic nerve following lysophosphatidylcholine-induced demyelination

Summary In vivo application of lysophosphatidylcholine (LPC) to rat sciatic nerve induces impaired hind leg movement within 2 days which is recovered by 6 days. Segmental demyelination was seen at 2 days after LPC application, and remyelination had barely started in a few axons by 6 days. Using sodium channel-specific monoclonal antibodies and immunofluorescence microscopy, we observed altered distribution of sodium channels in demyelinated axons. Bright fluorescent labeling was found along the segmentally demyelinated axolemma at 6 days in contrast to the dim staining of the demyelinated nerve found at 2 days. In addition, radioimmunoassays detected an elevated number of antibody binding sites on sciatic nerve trunk from the sixth day. Our data provide the immunocytochemical evidence for the assumption that recruitment of sodium channels into demyelinated axolemma contributes to the recovery of function following axon demyelination by LPC.     

Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury

Traumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects against ischemic brain injuries with anti-oxidant and anti-inflammatory properties, and by scavenging reactive species. However, whether caffeic acid has protective effects against traumatic brain injury is unknown. Therefore, we determined the effect of caffeic acid on the lesion in the early (1 day) and late phases (7 to 28 days) of cryoinjury in mice. We found that caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days, but attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and glial fibrillary acidic protein (GFAP) protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduction of superoxide dismutase activity and the increase in malondialdehyde content in the brain 1 day after cryoinjury. These results indicate that caffeic acid exerts a protective effect in traumatic brain injury, especially on glial scar formation in the late phase, which at least is associated with its anti-oxidant ability.     

Temporal Profile of Astrocytes and Changes of Oligodendrocyte-Based Myelin Following Middle Cerebral Artery Occlusion in Diabetic and Non-diabetic Rats

The long-term impacts of cerebral ischemia and diabetic ischemia on astrocytes and oligodendrocytes have not been defined. The objective of this study is to define profile of astrocyte and changes of myelin in diabetic and non-diabetic rats subjected to focal ischemia.Focal cerebral ischemia of 30-min duration was induced in streptozotocin-induced diabetic and vehicle-injected normoglycemic rats. The brains were harvested for immunohistochemistry of glial fibrillary acidic protein (GFAP) and 2'', 3''-cyclic nucleotide 3''-phosphodiesterase (CNPase) at various reperfusion endpoints ranging from 30 min up to 28 days. The results showed that activate astrocytes were observed after 30 min and peaked at 3 h to 1 day after reperfusion in ischemic penumbra, and peaked at 7 days of reperfusion in ischemic core. Diabetes inhibited the activation of astrocytes in ischemic hemisphere. Demyelination occurred after 30 min of reperfusion in ischemic core and peaked at 1 day. Diabetes caused more severe demyelination compared with non-diabetic rats. Remyelination started at 7 days and completed at 14 and 28 days in ischemic region. Diabetes inhibited the remyelination processes. It is concluded that ischemia activates astrocytes and induces demyelination. Diabetes inhibits the activation of astrocytes, exacerbates the demyelination and delays the remyelination processes. These may contribute to the detrimental effects of hyperglycemia on ischemic brain damage.     

Administration of simvastatin after kainic acid-induced status epilepticus restrains chronic temporal lobe epilepsy

In this study, we examined the effect of chronic administration of simvastatin immediately after status epilepticus (SE) on rat brain with temporal lobe epilepsy (TLE). First, we evaluated cytokines expression at 3 days post KA-lesion in hippocampus and found that simvastatin-treatment suppressed lesion-induced expression of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α). Further, we quantified reactive astrocytosis using glial fibrillary acidic protein (GFAP) staining and neuron loss using Nissl staining in hippocampus at 4-6 months after KA-lesion. We found that simvastatin suppressed reactive astrocytosis demonstrated by a significant decrease in GFAP-positive cells, and attenuated loss of pyramidal neurons in CA3 and interneurons in dentate hilar (DH). We next assessed aberrant mossy fiber sprouting (MFS) that is known to contribute to recurrence of spontaneous seizure in epileptic brain. In contrast to the robust MFS observed in saline-treated animals, the extent of MFS was restrained by simvastatin in epileptic rats. Attenuated MFS was related to decreased neuronal loss in CA3 and DH, which is possibly a mechanism underlying decreased hippocampal susceptibility in animal treated with simvastatin. Electronic encephalography (EEG) was recorded during 4 to 6 months after KA-lesion. The frequency of abnormal spikes in rats with simvastatin-treatment decreased significantly compared to the saline group. In summary, simvastatin treatment suppressed cytokines expression and reactive astrocytosis and decreased the frequency of discharges of epileptic brain, which might be due to the inhibition of MFS in DH. Our study suggests that simvastatin administration might be a possible intervention and promising strategy for preventing SE exacerbating to chronic epilepsy.     

Intralesional injection of interferon gamma affects reactive proliferation of astrocytes in the neonatal rat brain. A preliminary study of the age-dependent effect

Following a mechanical lesion of the left cerebral hemisphere, newborn male rats received a single injection of recombinant rat interferon gamma (IFN gamma) into the lesion cavity at doses of 5, 50 and 500 U. One or two days after the injury the rats were injected with 3H-thymidine. Brain sections were immunostained for glial fibrillary acidic protein (GFAP), subjected to autoradiography and examined microscopically to record proliferating GFAP-immunopositive (GFAP+) astrocytes labeled with 3H-thymidine. Following the intermediate 50 U dose of IFN gamma, numbers of GFAP+ astrocytes and of their mitoses on day 1 after injury were significantly higher than in controls. Nevertheless, the astrocyte labeling index remained at the control level. Injections of the minimal 5 U or the maximal 500 U doses of IFN gamma had no effect on that day. On day 2, however, each of the three doses evoked a statistically significant but dose-independent reduction of the labeling index without similar changes in total number of GFAP+ astrocytes or in numbers of their divisions. The changes appear to indicate a non-linear relation between the intensity of reactive behaviour of astrocytes and the amount of IFN gamma injected into the lesion area. On the basis of previous publications, IFN gamma effects on the astrocyte reactivity are discussed as being age-dependent.     

Effects of intracerebral administration of IL-1beta on reactive behaviors of astrocytes and macrophages in the injured brain of newborn rats

Newborn male rats were subjected to a mechanical lesion of the left cerebral hemisphere. Thereafter, a single dose 5 or 500 units (U) of recombinant rat interleukin-1beta (IL-1beta) was injected into the lesion cavity. One or 2 days after the injury, the rats were injected with 3H-thymidine to label dividing cells. Brain sections were subjected to GFAP immunocytochemistry or BSI-B4 lectin histochemistry to visualise astrocytes or macrophages, respectively. Autoradiography was used to detect cells proliferating within the region of injury in the immunocytochemically stained brain sections. The strongest mitogenic effect of IL-1beta on astrocytes (labeling index) was observed on day 1 after injury while a dose-dependent increase in their GFAP-immunoreactivity occurred on day 2. At 500U dose, IL-1beta significantly reduced infiltration of macrophages on posttraumatic days 1 and 2 but did not influence their proliferation. Thus, effects of IL-1beta on the occurrence of macrophages were opposite to those on the GFAP-immunoreactivity of astrocytes and their proliferation. It appears to suggest existence of different mechanisms controlling reactive behaviors of the two cell populations.     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

A comparison of the proliferative activity of astrocytes and astrocyte-like cells expressing vimentin in the injured mouse cerebral hemisphere.

After an unilateral injury of the cerebral hemisphere, 28 nice were injected with 3H thymidine at different intervals following the injury. Thereafter, the distribution of autographically labelled astrocytes expressing glial fibrillary protein (GFAP) and astrocyte-like cells expressing vimentin were recorded within the region of injury. Proliferative activity of the two cell types started at the same time, i.e. 24 h. after injury, reached its maximum on day 4, and returned to normal level after the 8th posttraumatic day. However, on day 4 the number of proliferating GFAP-positive astrocytes was about 50% higher than that of vimentin-positive cells. This was regarded as a proof of the concept that a significant number of astrocytes did not express vimentin during its mitotic cycle. Those facts were considered as an evidence against the hypothesis that a reactive astrocyte division induces a two-stage increase in the cytoskeletal proteins level with the elevated synthesis of vimentin preceding that of GFAP.     

Ibotenic acid-induced lesions of striatal target and projection neurons: ultrastructural manifestations in dopaminergic and non-dopaminergic neurons and in glia

The cytological changes elicited by central microinjections of the excitotoxin, ibotenic acid (IBO) were examined in the adult rat striatonigral system using electron microscopic immunocytochemistry. The chemical markers included tyrosine hydroxylase (TH), a biosynthetic enzyme in dopaminergic neurons, and glial fibrillary acidic protein (GFAP). Both short (1-7 day) and long (30-60 days) term effects were evaluated at the site of IBO-injections in the striatum and more distally in the substantia nigra, which both contributes afferents and receives efferents from the striatum. In the neostriatum at every survival period examined, TH-labeled axonal processes appeared equally numerous in the control and IBO-injected hemispheres. However, the TH-labeled axons in the striatum ipsilateral to the IBO-injection were slightly enlarged, and generally lacked synaptic densities. In the early period the remaining neuropil showed signs of edema and contained perikarya and dendrites with vacuolar or dense cytoplasm as well as intact, unlabeled terminals. Numerous astrocytes, and apparently demyelinated axons were more commonly seen at the 7 day period. At 30 and 60 days, bundles of myelinated axons, unlabeled axon terminals, and astrocytes containing a variety of cytosomes and other cytoplasmic inclusions were in close apposition to TH-labeled axon terminals. These results suggest that the dopaminergic terminals may serve neuromodulatory functions with respect to glia or other afferent axons remaining after IBO-injections in the striatum. In the substantia nigra, homolateral to the injection, a dense type of degeneration was seen in a few perikarya and dendrites at 7 days of survival. At this stage, electron dense anterograde degeneration also was seen in terminals contacting both TH-labeled and unlabeled dendrites. The secondary long term changes in nuclear groups located distal to the primary lesion are characteristic of certain types of progressive human neuropathological disorders.     

Astrocytosis and cathepsin D activity in experimental measles encephalomyelitis

The temporal relationship between the activity of cathepsin D (CD), the major brain acid proteinase, inflammatory cell infiltration and reactive astrocytosis was examined in a hamster model of measles virus infection of the central nervous system. Twenty-five day old hamsters were inoculated intracerebrally with the HBS strain of measles virus and sacrificed 6, 10, and 16 days later. Mean CD levels in aqueous extracts of infected brain were significantly elevated on days 10 and 16 compared to control animals. Histologic examination showed that while the degree of inflammatory cell infiltration did not correlate with the elevations in CD activity (r=.38), there was a correlation with the degree of astrocytosis (r=.995). This suggests that the increase in CD was due to astrocytic changes and not directly related to mononuclear inflammatory cell infiltration.Preliminary results presented at the 20th Annual meeting of the American Society of Neurochemistry, Chicago, IL, March 9, 1989     

Functional Recovery Occurs Even After Partial Remyelination of Axon-Meshed Median and Ulnar Nerves in Mice

Upper limb nerve injuries are common, and their treatment poses a challenge for physicians and surgeons. Experimental models help in minimum exploration of the functional characteristics of peripheral nerve injuries of forelimbs. This study was conducted to characterize the functional recovery (1, 3, 7, 10, 14, and 21 days) after median and ulnar nerve crush in mice and analyze the histological and biochemical markers of nerve regeneration (after 21 days). Sensory–functional impairments appeared after 1 day. The peripheral nerve morphology, the nerve structure, and the density of myelin proteins [myelin protein zero (P0) and peripheral myelin protein 22 (PMP22)] were analyzed after 21 days. Cold allodynia and fine motor coordination recovery occurred on the 10th day, and grip strength recovery was observed on the 14th day after injury. After 21 days, there was partial myelin sheath recovery. PMP22 recovery was complete, whereas P0 recovery was not. Results suggest that there is complete functional recovery even with partial remyelination of median and ulnar nerves in mice.

     

Laminin is induced in astrocytes of adult brain by injury.

Laminin is a high mol. wt. non-collagenous matrix glycoprotein, confined in adult tissues to basement membranes. In normal rat brain we found laminin mainly in vessel walls but, after injury, induced by stereotaxic injection of a neurotoxin, laminin immunoreactivity appeared also in reactive astrocytes, which are characteristically positive for the glial fibrillary acidic protein (GFAP). Laminin was first detected in GFAP-immunoreactive glial cells 24 h after injury. Four days later the majority of reactive astrocytes in the gray matter were positive for laminin and the laminin immunoreactivity, but not that of GFAP, gradually subsided within a month. Fibronectin, the other major matrix glycoprotein, was found only in capillary structures both in normal and lesioned brain tissue. The results indicate that mature astrocytes have the potential to produce laminin and suggest a role for this glycoprotein in brain regeneration.     

Oxidation and structural perturbation of redox-sensitive enzymes in injured skeletal muscle

Molecular events that control skeletal muscle injury and regeneration are poorly understood. However, inflammation associated with oxidative stress is considered a key player in modulating this process. To understand the consequences of oxidative stress associated with muscle injury, inflammation, and regeneration, hind-limb muscles of C57Bl/6J mice were studied after injection of cardiotoxin (CT). Within 1 day post-CT injection, polymorphonuclear neutrophilic leukocyte accumulation was extensive. Compared to baseline, tissue myeloperoxidase (MPO) activity was elevated eight- and fivefold at 1 and 7 days post-CT, respectively. Ubiquitinylated protein was elevated 1 day postinjury and returned to baseline by 21 days. Cysteine residues of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were irreversibly oxidized within 1 day post-CT injection and were associated with protein conformational changes that fully recovered after 21 days. Importantly, protein structural alterations occurred in conjunction with significant decreases in CK activity at 1, 3, and 7 days post-CT injury. Interestingly, elevations in tissue MPO activity paralleled the time course of conformational changes in CK and GAPDH. In combination, these results demonstrate that muscle proteins in vivo are structurally and functionally altered via the generation of reactive oxygen species produced during inflammatory events after muscle injury and preceding muscle regeneration.     

Multiple cryotherapy applications attenuate oxidative stress following skeletal muscle injury

Objectives: To investigate the effects of multiple cryotherapy applications after muscle injury on markers of oxidative stress.

Methods: Following cryolesion-induced skeletal muscle injury in rats, ice was applied at the injured site for 30?minutes, three times per day, on the day of injury, and for 2 days after injury. To determine the effect of the cryotherapy treatment on markers of oxidative stress, biochemical analyses were performed 3, 7, and 14 days after injury.

Results: Compared with non-treated animals, cryotherapy reduced dichlorofluorescein at 7 and 14 days post-injury and thiobarbituric acid reactive substances levels at 3 and 7 days post-injury (P?P?>?0.05), whereas non-treated groups demonstrated lower levels than the control group (P?P?P?=?0.92).

Discussion: Cryotherapy reduced the production of reactive oxygen species after muscle injury, resulting in an attenuated response of the antioxidant system. These findings suggest that using multiple cryotherapy applications is efficient to reduce oxidative stress.     

Axonal Regeneration after Sciatic Nerve Lesion Is Delayed but Complete in GFAP- and Vimentin-Deficient Mice

Peripheral axotomy of motoneurons triggers Wallerian degeneration of injured axons distal to the lesion, followed by axon regeneration. Centrally, axotomy induces loss of synapses (synaptic stripping) from the surface of lesioned motoneurons in the spinal cord. At the lesion site, reactive Schwann cells provide trophic support and guidance for outgrowing axons. The mechanisms of synaptic stripping remain elusive, but reactive astrocytes and microglia appear to be important in this process. We studied axonal regeneration and synaptic stripping of motoneurons after a sciatic nerve lesion in mice lacking the intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin, which are upregulated in reactive astrocytes and Schwann cells. Seven days after sciatic nerve transection, ultrastructural analysis of synaptic density on the somata of injured motoneurons revealed more remaining boutons covering injured somata in GFAP^–/–Vim^–/– mice. After sciatic nerve crush in GFAP^–/–Vim^–/– mice, the fraction of reinnervated motor endplates on muscle fibers of the gastrocnemius muscle was reduced 13 days after the injury, and axonal regeneration and functional recovery were delayed but complete. Thus, the absence of GFAP and vimentin in glial cells does not seem to affect the outcome after peripheral motoneuron injury but may have an important effect on the response dynamics.     

Developmental expression of glial markers in ependymocytes of the rat subcommissural organ: role of the environment

Summary The rat subcommissural organ (SCO), principally composed of modified ependymocytes (a type of glial cell), is a suitable model for the in vivo study of glial differentiation. An immunohistochemical study of the ontogenesis of rat SCO-ependymocytes from embryonic day 13 to postnatal day 10 shows that these cells express transitory glial fibrillary acidic protein (GFAP) from embryonic day 19 until postnatal day 3. However, S100 protein (S100) is never expressed in the SCO-cells, contrasting with the ventricle-lining cells of the third ventricle, which contain S100 as early as embryonic day 17. Environmental factors could be responsible for the repression of GFAP and S100 in adult rats, because GFAP and S100 are observed in ependymocytes of SCO 3 months after being grafted from newborn rat into the fourth ventricle of an adult rat. Neuronal factors might be involved in the control of the expression of S100, since after the destruction of serotonin innervation by neurotoxin at birth, S100 can be observed in some SCO-ependymocytes of adult rats. On the other hand, GFAP expression is apparently not affected by serotomin denervation, suggesting the existence of several factors involved in the differentiation of SCO-cells.