Heme oxygenase 1 in cerebrospinal fluid from infants and children after severe traumatic brain injury

Heme oxygenase 1 (HO-1) is an enzyme important in the catabolism of heme that is induced under conditions of oxidative stress. HO-1 degradation of heme yields biliverdin, bilirubin, carbon monoxide and iron. HO-1 is thought to serve a protective antioxidant function, and upregulation of HO-1 has been demonstrated in experimental models of neurodegeneration, subarachnoid hemorrhage, cerebral ischemia and traumatic brain injury (TBI). We measured HO-1 concentration in cerebral spinal fluid samples from 48 infants and children following TBI and 7 control patients by ELISA. Increased HO-1 was seen in TBI versus control patients--mean 2.75+/-0.63, peak 4.17+/-0.96 ng/ml versus control (<0.078 ng/ml, not detectable) (p<0.001). Increased HO-1 concentration was associated with increased injury severity and unfavorable neurological outcome (both p<0.05). Increased HO-1 concentration was independently associated with younger age; however, statistical analysis could not rule out the possibility that the effect of age was related to inflicted TBI from child abuse. HO-1 increases after TBI and appears to be more prominent in infants compared with older children after injury.     

Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat

The induction of heme oxygenase-1 (HO-1) was studied in intact spinal cords and injured spinal cords after a moderate, thoracic contusion injury. HO-1 was immunolocalized in the normal cord and along the axis of the cord at 1, 2, 3 and 4 days after contusion. Induction of this enzyme in astrocytes and microglia/macrophages was evaluated using immunofluorescent double labeling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor. HO-1 was expressed in neurons in the normal spinal cord. After contusion, HO-1 was induced in both gray and white matter at the impact site. In segments of cord that were 1 cm proximal or distal to the injury, HO-1 was primarily induced in the dorsal columns and occasionally in the lateral white matter. This pattern of induction was noted at all time points. The HO-1 was induced primarily in microglia/macrophages. The distribution of the HO-1 positive cells closely correlated with the pattern of intraparenchymal hemorrhage. These findings demonstrate acute induction of HO-1 in non-neuronal cells in the injured spinal cord. Induction of HO-1 in glia may be a consequence of multiple factors including exposure to heme proteins, hypoxia and oxidative stress.     

Long-term Induction of Haem Oxygenase-1 (HSP-32) in Astrocytes and Microglia Following Transient Focal Brain Ischaemia in the Rat

Haem oxygenase-1 (HO-1) is a stress protein and a rate-limiting enzyme in haem degradation, generating ferrous iron, carbon monoxide and bile pigments. HO-1 has been suggested to be protective against oxidative stress. In the normal rodent brain the enzyme is localized in selected neuron populations, but heat shock, glutathione depletion in vivo and oxidative stress in vitro induce HO-1 predominantly in glial cells. We studied HO-1 expression in the brain following transient occlusion of the middle cerebral artery, and found increased mRNA levels in the ischaemic region from 4 h to 7 days after 90 min of ischaemia. The mRNA levels peaked at 12 h, and were localized perifocally. HO-1-immunoreactive astrocytes and microglial cells were seen in the perifocal area, in the ipsilateral and occasionally in the contralateral hippocampus. Some perifocal neurons were also HO-14mmunoreactive. In the infarcted area HO-1-positive microglia/macrophages were detected in double-labelling experiments. A microassay measuring the conversion of [¹⁴C]haem to [¹⁴C]bilirubin showed a two-fold increase in haem oxygenase activity in the infarcted core. These observations show a long-term induction of HO-1 protein and its activity following ischaemia-reperfusion brain injury, and indicate increased capacity for haem degradation and the generation of biologically active bile products, carbon monoxide and iron in astrocytes and some microglia/macrophages during focal brain ischaemia.     

Sustained induction of heme oxygenase-1 in the traumatized spinal cord

Oxidative stress contributes to secondary injury after spinal cord trauma. Among the consequences of oxidative stress is the induction of heme oxygenase-1 (HO-1), an inducible isozyme that metabolizes heme to iron, biliverdin, and carbon monoxide. Here we examine the induction of HO-1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter. HO-1 was induced in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO-1 was induced in both the gray and the white matter. Thereafter, HO-1 expression was limited to degenerating fiber tracts. HSP70, a heat shock protein induced mainly by the presence of denatured proteins, was consistently colocalized with HO-1 in the microglia and macrophages. This study to demonstrates long-term induction of HO-1 and HSP70 in microglia and macrophages after traumatic injury and an association between induction of HO-1 and Wallerian degeneration. White matter degeneration is characterized by phagocytosis of cellular debris and remodeling of surviving tissue. This results in the metabolism, synthesis, and turnover of heme and heme proteins. Thus, sustained induction of HO-1 and HSP70 in microglia and macrophages suggests that tissue degeneration is an ongoing process, lasting 6 weeks and perhaps even longer.     

Lesion-associated accumulation of uPAR/CD87- expressing infiltrating granulocytes, activated microglial cells/macrophages and upregulation by endothelial cells following TBI and FCI in humans

Urokinase-type plasminogen activator receptor (uPAR/CD87) together with its ligand, urokinase-type plasminogen activator (uPA), constitutes a proteolytic system associated with tissue remodelling and leucocyte infiltration. uPAR is a member of the glycosyl phosphatidyl inositol (GPI) anchored protein family. The functional role of uPAR comprises fibrinolysis by conversion of plasminogen to plasmin. In addition, uPAR promotes cell adhesion, migration, proliferation, re-organization of the actin cytoskeleton, and angiogenesis. Furthermore, uPAR is involved in prevention of scar formation and is chemoattractant to macrophages and leucocytes. In order to investigate the pathophysiological role of uPAR following human CNS injury we examined necrotic brain lesions resulting from traumatic brain injury (TBI; n = 28) and focal cerebral infarctions (FCI; n = 17) by immunohistochemistry. Numbers of uPAR+ cells and uPAR+ blood vessels were counted. Following brain damage, uPAR+ cells increased significantly within 12 h, reached a maximum after 3-4 days and remained elevated until later stages. uPAR was expressed by infiltrating granulocytes, activated microglia/macrophages and endothelial cells. Numbers of uPAR+ vessels increased in parallel subsiding earlier following FCI than post TBI. The restricted, lesion-associated accumulation of uPAR+ cells in the brain parenchyma and upregulated expression by endothelial cells suggests a crucial role for the influx of inflammatory cells and blood-brain barrier (BBB) disturbance. Through a failure in BBB function, uPAR participates in formation of brain oedema and thus contributes to secondary brain damage. In conclusion, the study defines the localization, kinetic course and cellular source of uPAR as a potential pharmacological target following human TBI and FCI.     

Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage

The heme released following subarachnoid hemorrhage is metabolized by heme-oxygenase (HO) to biliverdin and carbon monoxide (CO) with the release of iron. The HO reaction is important since heme may contribute to vasospasm and increase oxidative stress in cells. HO is comprised of at least two isozymes, HO-2 and HO-1. HO-1, also known as heat shock protein HSP32, is inducible by many factors including heme and heat shock. HO-2 does not respond to these stresses. To begin to examine HO activity following subarachnoid hemorrhage (SAH), the expression of HO-1 and HO-2 was investigated after experimental SAH in adult rats. Immunocytochemistry for HO-1, HO-2 and HSP70 proteins was performed at 1, 2, 3 and 4 days after injections of lysed blood, whole blood, oxyhemoglobin and saline into the cisterna magna. A large increase in HO-1 immunoreactivity was seen in cells throughout brain following injections of lysed blood, whole blood, and oxyhemoglobin but not saline. Lysed blood, whole blood and oxyhemoglobin induced HO-1 in all of the cortex, hippocampus, striatum, thalamus, forebrain white matter and in cerebellar cortex. HO-1 immunoreactivity was greatest in those regions adjacent to the basal subarachnoid cisterns where blood and oxyhemoglobin concentrations were likely highest. Double immunofluorescence studies showed the HO-1 positive cells to be predominately microglia, though HO-1 was induced in some astrocytes. HO-1 expression resolved by 48 h. HO-2 immunoreactivity was abundant but did not change following injections of blood. A generalized induction of HSP70 heat shock protein was not observed following injections of lysed blood, whole blood, oxyhemoglobin, or saline. These results suggest that HO-1 is induced in microglia throughout rat brain as a general, parenchymal response to the presence of oxyhemoglobin in the subarachnoid space and not as a stress response. This microglial HO-1 response could be protective against the lipid peroxidation and vasospasm induced by hemoglobin, by increasing heme clearance and iron sequestration, and enhancing the production of the antioxidant bilirubin.     

Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of lysed blood

The initial aim of this study was to determine if the HSP70 (the main inducible heat shock protein), HO-1 (heme oxygenase-1, HSP32) and HSP47 (a collagen chaperone) stress proteins were induced in the same focal regions of rat brain following experimental subarachnoid hemorrhage (SAH). The next objective was to determine whether anti-oxidants prevented the stress gene expression in the focal regions. Lysed blood (150 microliter) was injected into the subarachnoid space of adult, female Sprague-Dawley rats via the cisterna magna. Animals were sacrificed 24 h later. Immunocytochemistry showed focal regions of stress gene induction in most animals (13/21), HSP70 and HO-1 proteins being expressed in neurons, microglia and astrocytes and HSP47 being expressed in microglia. Co-induction of the same three stress proteins was observed in focal areas in the striatum and cerebellum as well. In the 13 animals with focal regions of stress gene induction there were 8.1+/-1.8 foci in cortex, 5.5+/-0.9 foci in striatum, and 11.7+/-7.3 foci in cerebellum in the brain of each animal. The focal regions of stress gene induction varied in size from 200 micrometer to 7 mm in diameter. Systemic administration of the tirilazad-like anti-oxidants U101033E (n=8) and U74389G (n=7) completely blocked stress protein induction in focal brain regions normally produced by cisternal injections of lysed blood. There were fewer drug treated animals (0/15) with focal areas of stress gene induction compared to non-drug (13/21) treated animals following the cisternal lysed blood injections (p<0.01 using Fisher's probability test). This study shows that anti-oxidants prevent focal regions of injury as assessed by heat shock protein expression in a rat model of SAH.     

N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury

Traumatic brain injury (TBI) results in a cascade of events that includes the production of reactive oxygen species. Heme oxygenase-1 (HO-1) is induced in glial cells following head trauma, suggestive of oxidative stress. We have studied the temporal and spatial effects of the antioxidant N-acetylcysteine (NAC) on HO-1 levels following lateral fluid-percussion injury by immunoblotting and immunohistochemistry. In the injured cerebral cortex, maximal HO-1 induction was seen 6 h post-TBI and was maintained for up to 24 h following the insult, while the ipsilateral hippocampus and thalamus showed marked induction at 24 h postinjury. In all three brain regions, little or no HO-1 immunoreactivity was observed on the contralateral side. Astrocytes exhibited positive immunoreactivity for HO-1 in the injured cerebral cortex, hippocampus, and thalamus, while some neurons and microglia were also immunoreactive in the injured cortex. The administration of NAC 5 min following TBI resulted in a marked reduction in this widespread induction of HO-1, concomitant with a decrease in the volume of injury in all three brain regions. Together, these findings indicate that HO-1 induction is related to both oxidative and injury characteristics of the affected tissue, suggesting that protein expression of this gene is a credible marker of oxidative damage in this model of TBI.     

Microglia and macrophages are major sources of locally produced transforming growth factor-β1 after transient middle cerebral artery occlusion in rats

The potentially neurotrophic cytokine transforming growth factor-β₁ (TGF-β₁) is locally expressed following human stroke and experimental ischemic lesions, but the cellular source(s) and profile of induction have so far not been established in experimental focal cerebral ischemia. This study presents the time course and a cellular localization of TGF-β₁ mRNA, visualized by in situ hybridization combined with immunohistochemical staining for microglia, macrophages, or astrocytes, on brain sections from adult spontaneously hypertensive rats subjected to transient proximal occlusion of their middle cerebral artery. Six hours after ischemia, an early and transient neuronal and microglial expression of TGF-β₁ mRNA was observed in the extraischemic cingulate and frontal cortices. Both early and protracted expression of TGF-β₁ mRNA in the caudate-putamen and neocortical infarcts and in the caudate-putamen penumbra colocalized with OX42/ ED1-immunoreactive microglia and macrophages, whereas TGF-β₁ mRNA in the neocortical penumbra colocalized with OX42/ ED1-immunoreactive cells of a microglial morphology. No astrocytes were double-labeled. The number of TGF-β₁ mRNA-expressing microglia and macrophages increased strongly during the first week. Thereafter, TGF-β₁ mRNA became increasingly restricted to the neocortical penumbra (3 weeks), and after 3 months it was confined to activated microglia in the anterior commisure. Our data establish activated microglia and macrophages as the major source of TGF-β₁ mRNA following experimental focal cerebral ischemia. Consequently, TGF-β₁-mediated functions may be exerted by microglia both in the early degenerative phase, and later in combination with blood-borne macrophages, in the remodeling and healing phase after focal cerebral ischemia. GLIA 24:437–448, 1998. © 1998 Wiley-Liss, Inc.     

Infiltrating CD14+ monocytes and expression of CD14 by activated parenchymal microglia/macrophages contribute to the pool of CD14+ cells in ischemic brain lesions

CD14, a key pattern recognition receptor of the innate immune system, is a surface molecule on monocytic cells involved in cellular activation. We investigated 18 autopsy cases of focal cerebral infarctions (FCI) by immunohistochemistry to examine CD14 expression following ischemia. Controls confirmed constitutive CD14 expression by few perivascular cells. In contrast to quiescent CD14- parenchymal microglial cells, following ischemia activated microglia/macrophages expressed abundant CD14. In FCI, CD14+ cells increased both in perivascular spaces and in brain parenchyma within 1-2.5 days and remained elevated until late stages. Early CD14 expression suggests an essential part of CD14 in the acute inflammatory response following stroke.     

Selective accumulation of cyclooxygenase-1-expressing microglial cells/macrophages in lesions of human focal cerebral ischemia

Cyclooxygenases (COX; prostaglandin endoperoxide H synthases) are key enzymes in the conversion of arachidonic acid into prostanoids which mediate inflammation, immunomodulation, mitogenesis, ovulation, fewer, apoptosis and blood flow. Here, we report COX-1 expression following focal cerebral infarctions (FCI). In healthy control brains, COX-1 was localized by immunohistochemistry to a few endothelial cells, single neurons and rare, evenly distributed brain microglia/macrophages. In infarctioned brains, COX-1+ cells accumulated highly significantly (P < 0.0001) in peri-infarctional areas and in the developing necrotic core early after infarction. Here, cell numbers remained persistently elevated up to several months post infarction. Further, clusters of COX-1+ cells were located in perivascular regions related to the Virchow-Robin space. Double-labeling experiments confirmed co-expression of COX-1 by CD68+ microglia/macrophages. Co-expression of the activation antigens HLA-DR, -DP, -DQ (MHC class II) or the macrophage inhibitor factor-related protein MRP-8 (S100A8) by most COX-1+ microglia/macrophages was only seen early after infarction. Thus, COX-1 appeared to be expressed in microglial cells regardless of their activation state. However, the prolonged accumulation of COX-1+ microglia/macrophages restricted to peri-infarctional areas enduring the acute post-ischemic inflammatory response points to a role of COX-1 in tissue remodeling or in the pathophysiology of secondary injury. We have identified localized, accumulated COX-1 expression as a potential pharmacological target following FCI. Therefore we suggest that therapeutic approaches based on selective COX-2 blocking might not be sufficient for suppressing the local synthesis of prostanoids.     

Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier

The induction of the stress protein heme oxygenase-1 (HO-1) was studied in the rat brain after intracarotid administration of hyperosmolar mannitol. HO-1 was immunolocalized in fixed sections of brain 24 h to 7 days after injection. Immunoglobulin G (IgG) was immunolocalized in adjacent sections to demonstrate areas of breakdown of the blood–brain barrier. Induction of HO-1 was also evaluated by Western immunoblots, performed at 24 h after the insult. Immunofluorescent double labelling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor was used to determine if glia/macrophages expressed HO-1. There was pronounced, widespread induction of HO-1 in the ipsilateral hemisphere and cerebellum by 24 h both by immunocytochemistry and by Western blots. This induction was markedly attenuated at later times. HO-1 was induced in astrocytes and microglia/macrophages in the ipsilateral hemisphere. In addition, the protein was induced in Bergmann glia and scattered microglia/macrophages in the cerebellum. The mechanism of induction of HO-1 in glia after opening of the blood–brain barrier could include exposure to heme proteins, denatured proteins and other plasma constituents known to induce HO-1. This glial induction may reflect a protective response of these cells.     

In vitro and in vivo induction of heme oxygenase-1 in rat glial cells: Possible involvement of nitric oxide production from inducible nitric oxide synthase

To determine whether heme oxygenase-1 (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-γ (IFN-γ), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO₂⁻accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as N^G-nitro-L-arginine (NNA) and N^G-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO₂⁻ accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-γ, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-γ. These results suggest that endogenous NO production by iNOS in microglia causes autocrine and paracrine induction of HO-1 protein in microglia and astrocytes in vitro and in rat brain. GLIA 22:138–148, 1998.© 1998 Wiley-Liss, Inc.     

Acute intranasal osteopontin treatment in male rats following TBI increases the number of activated microglia but does not alter lesion characteristics

Intranasal recombinant osteopontin (OPN) has been shown to be neuroprotective in different models of acquired brain injury but has never been tested after traumatic brain injury (TBI). We used a model of moderate-to-severe controlled cortical impact in male adult Sprague Dawley rats and tested our hypothesis that OPN treatment would improve neurological outcomes, lesion and brain tissue characteristics, neuroinflammation, and vascular characteristics at 1 day post-injury. Intranasal OPN administered 1 hr after the TBI did not improve neurological score, lesion volumes, blood–brain barrier, or vascular characteristics. When assessing neuroinflammation, we did not observe any effect of OPN on the astrocyte reactivity but discovered an increased number of activated microglia within the ipsilateral hemisphere. Moreover, we found a correlation between edema and heme oxygenase-1 (HO-1) expression which was decreased in OPN-treated animals, suggesting an effect of OPN on the HO-1 response to injury. Thus, OPN may increase or accelerate the microglial response after TBI, and early response of HO-1 in modulating edema formation may limit the secondary consequences of TBI at later time points. Additional experiments and at longer time points are needed to determine if intranasal OPN could potentially be used as a treatment after TBI where it might be beneficial by activating protective signaling pathways.     

Traumatic brain injury leads to increased expression of peripheral-type benzodiazepine receptors, neuronal death, and activation of astrocytes and microglia in rat thalamus

In mammalian CNS, the peripheral-type benzodiazepine receptor (PTBR) is localized on the outer mitochondrial membrane within the astrocytes and microglia. PTBR transports cholesterol to the site of neurosteroid biosynthesis. Several neurodegenerative disorders were reported to be associated with increased densities of PTBR. In the present study, we evaluated the changes in the PTBR density and gene expression in the brains of rats as a function of time (6 h to 14 days) after traumatic brain injury (TBI). Sham-operated rats served as control. Between 3 and 14 days after TBI, there was a significant increased in the binding of PTBR antagonist [(3)H]PK11195 (by 106 to 185%, P < 0.01, as assessed by quantitative autoradiography and in vitro filtration binding) and PTBR mRNA expression (by 2- to 3. 4-fold, P < 0.01, as assessed by RT-PCR) in the ipsilateral thalamus. At 14 days after the injury, the neuronal number decreased significantly (by 85 to 90%, P < 0.01) in the ipsilateral thalamus. At the same time point, the ipsilateral thalamus also showed increased numbers of the glial fibrillary acidic protein positive cells (astrocytes, by approximately 3.5-fold) and the ED-1 positive cells (microglia/macrophages, by approximately 36-fold), the two cell types known to be associated with PTBR. Increased PTBR expression following TBI seems to be associated with microglia/macrophages than astrocytes as PTBR density at different periods after TBI correlated better with the number of ED-1 positive cells (r(2) = 0.95) than the GFAP positive cells (r(2) = 0.56). TBI-induced increased PTBR expression is possibly an adaptive response to cellular injury and may play a role in the pathophysiology of TBI.     

血红素氧合酶-1及血红素氧合酶-2在大鼠局灶性脑缺血中的意义

目的　研究血红素氧合酶 1(HO 1)及血红素氧合酶 2 (HO 2 )在局灶性脑缺血中的作用。方法　采用大鼠大脑中动脉栓塞脑缺血模型 ,对 6 6只大鼠脑缺血后不同时间点进行HO 1、HO 2免疫组化染色及病理学研究 ,并用计算机图像分析技术计算两者表达水平。结果　栓塞后 30min大鼠皮质及海马即有HO 1阳性神经元及胶质细胞的表达 ,且随着时间推移HO 1的表达逐渐增强 ,到栓塞后 12h达峰值 (P <0 0 1) ,以后逐渐下降 ,栓塞后 1周仍有HO 1表达。HO 2在正常大鼠及梗死大鼠脑组织内均有表达。栓塞后不同时间段 ,HO 2阳性神经元的数量无明显变化 (P >0 0 5 ) ,但HO 2表达呈动态变化 ,2 4h时最高 (P <0 0 1) ,以后逐渐下降。结论　脑缺血时脑内HO 1、HO 2表达的不同变化 ,是脑组织对损伤恢复重要的机制之一。HO 1修复受损的神经元和胶质细胞 ,而HO 2在于维护正常细胞的稳定     

Neurogenesis and glial proliferation are stimulated following diffuse traumatic brain injury in adult rats

Although increased neurogenesis has been described in rodent models of focal traumatic brain injury (TBI), the neurogenic response occurring after diffuse TBI uncomplicated by focal injury has not been examined to date, despite the pervasiveness of this distinct type of brain injury in the TBI patient population. Here we characterize multiple stages of neurogenesis following a traumatic axonal injury (TAI) model of diffuse TBI as well as the proliferative response of glial cells. TAI was induced in adult rats using an impact-acceleration model, and 5-bromo-2'-deoxyuridine (BrdU) was administered on days 1-4 posttrauma or sham operation to label mitotic cells. Using immunohistochemistry for BrdU combined with phenotype-specific markers, we found that proliferation was increased following TAI in the subventricular zone of the lateral ventricles and in the hippocampal subgranular zone, although the ultimate production of new dentate granule neurons at 8 weeks was not significantly enhanced. Also, abundant proliferating and reactive astrocytes, microglia, and polydendrocytes were detected throughout the brain following TAI, indicating that a robust glial response occurs in this model, although very few new cells in the nonneurogenic brain regions became mature neurons. We conclude that diffuse brain injury stimulates early stages of a neurogenic response similar to that described for models of focal TBI.     

Cultured astrocytes from heme oxygenase-1 knockout mice are more vulnerable to heme-mediated oxidative injury

Hemin, the oxidized form of heme, is released from hemoglobin after CNS hemorrhage and may contribute to injury to surrounding tissue. The heme oxygenase (HO) enzymes catalyze the breakdown of hemin to biliverdin, carbon monoxide, and ferric iron. Although HO-2, the isoform expressed predominantly in neurons, accelerates heme-mediated neuronal injury, inhibitor studies suggest that HO-1 induction has a protective effect on astrocytes. In the present study, we directly compared the vulnerability of cultured HO-1 knockout and wild-type astrocytes to hemin. Consistent with prior observations, exposure of wild-type cultures to hemin for 24 hr resulted in protein carbonylation and concentration-dependent cell death between 10 and 60 microM, as determined by MTT and lactate dehydrogenase release assays. In cultures prepared from mice lacking the HO-1 gene, oxidative cell injury was approximately doubled. Both protein oxidation and cell death in HO-1 knockout astrocytes were significantly reduced by pretreating cultures with an adenovirus encoding the HO-1 gene prior to hemin exposure. HO-2 expression was observed in both knockout and wild-type cultures and was not altered by HO-1 gene deletion. Cell hemin accumulation after 20 hr hemin exposure was 4.7-fold higher in knockout cells. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Selectively increasing its expression in astrocytes may be beneficial after hemorrhagic CNS injuries.     

Exacerbated glial response in the aged mouse hippocampus following controlled cortical impact injury

Old age is associated with enhanced susceptibility to and poor recovery from brain injury. An exacerbated microglial and astrocyte response to brain injury might be involved in poor outcomes observed in the elderly. The present study was therefore designed to quantitate the expression of markers of microglia and astrocyte activation using real-time RT-PCR, immunoblot and immunohistochemical analysis in aging brain in response to brain injury. We examined the hippocampus, a region that undergoes secondary neuron death, in aged (21–24 months) and adult (5–6 months) mice following controlled cortical impact (CCI) injury to the sensorimotor cortex. Basal mRNA expression of CD11b and Iba1, markers of activated microglia, was higher in aged hippocampus as compared to the adult. The mRNA expression of microglial markers increased and reached maximum 3 days post-injury in both adult and aged mice, but was higher in the aged mice at all time points studied, and in the aged mice the return to baseline levels was delayed. Basal mRNA expression of GFAP and S100B, markers of activated astrocytes, was higher in aged mice. Both markers increased and reached maximum 7 days post-injury. The mRNA expression of astrocyte markers returned to near basal levels rapidly after injury in the adult mice, whereas again in the aged mice return to baseline was delayed. Immunochemical analysis using Iba1 and GFAP antibodies indicated accentuated glial responses in the aged hippocampus after injury. The pronounced and prolonged activation of microglia and astrocytes in hippocampus may contribute to worse cognitive outcomes in the elderly following TBI.