Biomedical subjects

Staffan Holmin

Publications and source records attributed to Staffan Holmin.

15 recordsLinked to original sources

PubMed2006

Sustained survival of xenografted human neural stem/progenitor cells in experimental brain trauma despite discontinuation of immunosuppression.

Neural stem cells have emerged as a promising therapeutic tool in CNS disease and injuries. In the clinical setting, cultured human neural stem/progenitor cells (hNSC) are an attractive possibility for transplantation to the damaged brain. However, transplantation of hNSC requires toxic immunosuppressive treatment to avoid rejection. The aim of the current study was to evaluate if shortening the duration of immunosuppression by cyclosporin A would affect hNSC survival and differentiation after transplantation to the site of a focal brain injury in the rat. hNSC were xenografted to the hippocampus and the medial limit of an experimentally induced cortical contusion. The animals received immunosuppression for either 6 or 3 weeks or no immunosuppression. The status of the grafted human cells was analysed by immunohistochemistry. No statistically significant differences were observed between the two immunosuppressed groups regarding graft survival, migration or proliferation at 6 weeks post-transplantation. In contrast, the graft survival was extremely poor in the non-immunosuppressed group. Furthermore, the expression of the differentiation markers nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) in the transplanted cells did not differ significantly between the two immunosuppressed groups. Moreover, a fourth group of eight animals that were immunosuppressed for 3 weeks were allowed to survive for 6 months. Five of these rats demonstrated robust graft survival in the hippocampus and scattered cells in the cortex. This study demonstrates the importance of immunosuppression but also the possibility of shortening immunosuppression without impacting on the phenotype of the grafted hNSC.

Animals↗

PubMed2006

Neuroprotection by selective inhibition of inducible nitric oxide synthase after experimental brain contusion.

The inflammatory response is thought to be important for secondary damage following traumatic brain injury (TBI). The inducible nitric oxide synthase (iNOS) isoform is a mediator in inflammatory reactions and may catalyze substantial synthesis of NO in the injured brain. This study was undertaken to analyze neuronal degeneration and survival, cellular apoptosis and formation of nitrotyrosine following treatment with the iNOS-inhibitor L-N-iminoethyl-lysine (L-NIL) in a model of brain contusion. A brain contusion was produced using a weight-drop device in 30 rats. The animals received treatment with L-NIL or NaCl at 15 min and 12 h after the injury and were sacrificed at 24 h or 6 days after trauma. iNOS activity was measured at 24 h post-trauma by the conversion of L-[U- ( 14 )C]arginine to L-[U-( 14 )C]citrulline and immunohistochemistry for iNOS. Peroxynitrite formation was indirectly assessed by nitrotyrosine (NT) immunohistochemistry. Neuronal degeneration and survival were assessed by Fluoro-Jade (FJ) and NeuN stainings, and cellular death by TUNEL staining. iNOS activity but not iNOS immunoreactivity was significantly reduced in animals that received L-NIL. Neuronal degeneration (FJ) and NT immunoreactivity were significantly reduced at 24 h. Neuronal survival was unchanged at 24 h but increased at 6 days in L-NIL-treated animals. Cellular apoptosis of ED-1 and NeuN positive cells was significantly reduced following L-NIL treatment at 6 days after trauma. We demonstrated neuroprotection by selective inhibition of iNOS after trauma. L-NIL appeared to protect the injured brain by limiting peroxynitrite formation. Our findings support a putative harmful role of iNOS induction early after TBI.

Animals↗

PubMed2006

Inhibition of vascular endothelial growth factor receptor 2 activity in experimental brain contusions aggravates injury outcome and leads to early increased neuronal and glial degeneration.

Angiogenesis following traumatic brain injuries (TBIs) may be of importance for post-traumatic reparative processes and the development of secondary injuries. We have previously shown expression of vascular endothelial growth factor (VEGF), a major regulator of endothelial cell proliferation, angiogenesis and vascular permeability, and VEGF receptors (VEGFR1 and 2) after TBI in rat. In the present work we tried to further elucidate the role of VEGF after TBI by performing specific VEGFR2 activity inhibition. In rats subjected to VEGFR2 blockage we report an increased haemorrhagic area (P < 0.05), early increase in serum levels of neural injury marker neuron-specific enolase (P < 0.05) and glial injury marker S100beta (P < 0.05), and increased numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling- (TUNEL-) and FluoroJade B- (P < 0.05) positive cells, all increases preceding the known VEGF/VEGFR vascular response in brain trauma. An increase in lesion area, as measured by decreased microtubuli-associated protein 2 expression (P < 0.05) and increased glial fibrillary acidic protein reactivity (P < 0.05), could also be demonstrated. In addition, vascular density, as measured by von Willebrandt factor-positive cells, was decreased (P < 0.05). No differences in post-traumatic inflammatory response, as measured by stainings for macrophages, granulocytes and intracellular adhesion molecules, were shown between the groups. Taken together, our findings point towards VEGF/VEGFR2 up-regulation after TBI as being an important endogenous cytoprotective mechanism in TBI. The possible importance of VEGF on the vascular, neuronal and glial compartments of the neurovascular unit after TBI is discussed.

Angiogenesis Inhibitors↗

PubMed2005

Genomic responses in rat cerebral cortex after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) initiates a complex sequence of destructive and neuroprotective cellular responses. The initial mechanical injury is followed by an extended time period of secondary brain damage. Due to the complicated pathological picture a better understanding of the molecular events occurring during this secondary phase of injury is needed. This study was aimed at analysing gene expression patterns following cerebral cortical contusion in rat using high throughput microarray technology with the goal of identifying genes involved in an early and in a more delayed phase of trauma, as genomic responses behind secondary mechanisms likely are time-dependent. RESULTS: Among the upregulated genes 1 day post injury, were transcription factors and genes involved in metabolism, e.g. STAT-3, C/EBP-delta and cytochrome p450. At 4 days post injury we observed increased gene expression of inflammatory factors, proteases and their inhibitors, like cathepsins, alpha-2-macroglobulin and C1q. Notably, genes with biological function clustered to immune response were significantly upregulated 4 days after injury, which was not found following 1 day. Osteopontin and one of its receptors, CD-44, were both upregulated showing a local mRNA- and immunoreactivity pattern in and around the injury site. Fewer genes had decreased expression both 1 and 4 days post injury and included genes implicated in transport, metabolism, signalling, and extra cellular matrix formation, e.g. vitronectin, neuroserpin and angiotensinogen. CONCLUSION: The different patterns of gene expression, with little overlap in genes, 1 and 4 days post injury showed time dependence in genomic responses to trauma. An early induction of factors involved in transcription could lead to the later inflammatory response with strongly upregulated CD-44 and osteopontin expression. An increased knowledge of genes regulating the pathological mechanisms in trauma will help to find future treatment targets. Since trauma is a risk factor for development of neurodegenerative disease, this knowledge may also reduce late negative effects.

Animals↗

PubMed2005

Experimental subarachnoid hemorrhage induces changes in the levels of hippocampal NMDA receptor subunit mRNA.

NMDA receptors may play a crucial role in nerve cell death following subarachnoid hemorrhage (SAH). Changes in NMDA receptor-mediated transmission appear before neuronal death in rodent models of transient ischemia, and NMDA receptor function is known to be dependent on subunit composition. Here, we have investigated whether mRNA expression of the NMDA receptor subunits is altered in the hippocampal formation 3-5 h following experimental SAH, and correlated these early alterations to subsequent delayed cell death. SAH was induced by intraluminal perforation of the internal carotid artery intracranially, and cerebral blood flow (CBF) was bilaterally monitored by laser-Doppler flowmetry. Early changes in NMDA receptor subunit mRNA and early nerve cell death were analyzed at 3-5 h after SAH, and delayed nerve cell death was analyzed at 2-7 days after SAH. Duration of ipsilateral CBF reduction below 30% of baseline (CBF30) was predictive of ipsilateral delayed nerve cell death in the CA1 2-7 days after SAH. At CBF30 > 9 min, we found downregulation of mRNA for NR2A, NR2B, and NR3B at 3-5 h after SAH, whereas the levels of NR1 mRNA were unaffected. The downregulation of NR2A and NR2B mRNA may result in a reduced NMDA receptor function. Such reduction may be sufficient to provide neuroprotection in the dentate gyrus, where no cell death appears, but insufficient to rescue neurons in the hippocampus proper following SAH.

Animals↗

PubMed2005

VEGF and VEGF receptor expression after experimental brain contusion in rat.

Angiogenesis following traumatic brain injury (TBI) may be of importance not only for post-traumatic reparative processes but also for the development of secondary injuries. Vascular endothelial growth factor (VEGF) is a major regulator of endothelial cell proliferation, angiogenesis, and vascular permeability, though its possible involvement in secondary injuries after TBI is largely unknown. This study was undertaken to analyze the expression of VEGF and the VEGF receptors in experimental brain contusion in rat. Twenty-three adult female Sprague-Dawley rats were subjected to a focal cerebral contusion injury by use of a weight-drop model. Four additional rats underwent craniotomy only. The animals were sacrificed 6 h, or 1, 2, 4, 6, 8, or 16 days post-injury. Expression of VEGF and the VEGF receptors VEGFR1 (Flt-1) and VEGFR2 (Flk-1) were studied by in situ hybridization and immunohistochemistry. VEGF messenger (m)RNA and protein expression were detected in astrocytes, neutrophils, and macrophages in or adjacent to the injury from 1 day after injury, with a peak expression after 4-6 days. Flt-1 and Flk-1 mRNA and protein were detected in vessels adjacent to the lesion from 1 day after injury throughout day 6 after injury. It was also noted that Flt-1/Flk-1 and VEGF-positive vessels often were negative for SMI-71, a marker for vessels in areas with blood-brain barrier (BBB). In conclusion, we have demonstrated that TBI leads to an upregulation of VEGF, Flt-1, and Flk-1 mRNA and protein in and around the lesion. The data provide a foundation for future pharmacological intervention studies focusing on posttraumatic angiogenesis and possible injury repair effects of the VEGF system in TBI.

Animals↗

PubMed2005

Reduced neuronal injury after treatment with NG-nitro-L-arginine methyl ester (L-NAME) or 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) following experimental brain contusion.

OBJECTIVE: Nitric oxide (NO) and oxygen free radicals are implicated in the pathophysiology of traumatic brain injury (TBI). Peroxynitrite formation from NO and superoxide contributes to secondary neuronal injury but the neuroprotective effects of nitric oxide synthase (NOS)-inhibitors have been contradictory. This study was undertaken to examine whether PTtic administration of the (NOS)-inhibitor N-nitro-l-arginine methyl ester (L-NAME), and a combination of L-NAME and the nitrone radical scavenger 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) favorable affects neuronal injury in a model of TBI. METHODS: A weight-drop model of TBI was used. The animals received L-NAME, S-PBN or a combination of the drugs 15 minutes prothrombin time (PT) and sacrificed after 24 hours or six days. NOS activity was measured by the conversion of L-[U-C]arginine to L-[U-C]citrulline. Peroxynitrite formation, cellular apoptosis, neuronal degeneration and survival were assessed by nitrotyrosine-, TUNEL-, Fluoro-Jade- and NeuN-stainings. RESULTS: eNOS and nNOS activity was significantly reduced in animals that received L-NAME alone or the combination with S-PBN. iNOS activity or iNOS immunoreactivity was not affected. All treatments significantly reduced neuronal degeneration and nitrotyrosine immunoreactivity at 24 hours and increased neuronal survival at six days PT. No differences were detected between L-NAME and L-NAME + S-PBN groups. CONCLUSION: NO from NOS contributes to secondary neuronal injury in this TBI-model. PTtic treatment does not inhibit early beneficial NO-related effects. L-NAME and S-PBN limit peroxynitrite formation, promoting neuronal survival. The combination of L-NAME and S-PBN was neuroprotective; surprisingly no additive effects were found on nitrotyrosine formation, apoptosis or neuronal survival.

Animals↗

PubMed2004

In situ detection of intracerebral cytokine expression after human brain contusion.

The study was undertaken to analyze intracerebral expression of pro- and anti-inflammatory cytokines after traumatic brain injury (TBI) in man in order to compare the findings with previous experimental data regarding the pathogenesis of secondary brain injury. Contused brain tissue biopsies were obtained from 12 consecutive patients undergoing surgery for brain contusions 3 h to 5 days after trauma. Cytokine expression was analyzed by in situ hybridization and immunohistochemistry. In patients undergoing surgery less than 24 h after trauma, strong expression of both the pro-inflammatory cytokines interleukin (IL)-1-beta, IL-6 and interferon (IFN)-gamma and the anti-inflammatory cytokine IL-4 was detected. In patients undergoing surgery between 3 and 5 days after trauma, IL-4 expression was significantly lower (P < 0.05) compared to the patients operated early. IL-1-beta and IFN-gamma expression remained strong in comparison to IL-6 and IL-4 expression (P < 0.05). Immunohistochemistry for IL-1-beta confirmed that the protein was produced with a temporal and regional pattern that corresponded to in situ hybridization results. The study provides in situ data on intracerebral cytokine expression after contusion in the clinical setting. Strong intracerebral cytokine expression occurs in the perilesional zone both in the early and the delayed phase after traumatic brain injury in humans. The temporal regulation of pro- and anti-inflammatory cytokines differs which reveals different therapeutic windows for pharmacological intervention.

Adolescent↗

PubMed2004

MHC expression after human neural stem cell transplantation to brain contused rats.

Human neural stem cells survive and improve motor function after transplantation to the contused brain. However, the transplants might be rejected and that depends on the graft immunogenicity, the host immunological status and the immunosuppression strategy. We transplanted human neural stem cells to rats with brain contusion and analyzed the donor and host MHC antigen expression and the effect of a short-term immunosuppression with cyclosporine. In vitro human neural stem cells expressed only MHC-II antigens. This expression was down-regulated 6 weeks after transplantation. The host response was characterized by an increased MHC-II expression which was down-regulated by a longer term of immunosuppression. These findings are novel and necessary in order to understand the immunogenicity of human neural stem cell grafts.

Animals↗

PubMed2004

Proliferation, migration, and differentiation of human neural stem/progenitor cells after transplantation into a rat model of traumatic brain injury.

OBJECT: Cultures containing human neural stem and progenitor cells (neurospheres) have the capacity to proliferate and differentiate into the major phenotypes of the adult brain. These properties make them candidates for therapeutic transplantation in cases of neurological diseases that involve cell loss. In this study, long-term cultured and cryopreserved cells were transplanted into the traumatically injured rat brain to evaluate the potential for human neural stem/progenitor cells to survive and differentiate following traumatic injury. METHODS: Neural stem/progenitor cell cultures were established from 10-week-old human forebrain. Immunosuppressed adult rats received a unilateral parietal cortical contusion injury, which was delivered using the weight-drop method. Immediately following the injury, these animals received transplants of neural stem/progenitor cells, which were placed close to the site of injury. Two or 6 weeks after the procedure, these animals were killed and their brains were examined by immunohistochemical analysis. At both 2 and 6 weeks postoperatively, the transplanted human cells were found in the perilesional zone, hippocampus, corpus callosum, and ipsilateral subependymal zone of the rats. Compared with the 2-week time point, an increased number of HuN-positive cells was observed at 6 weeks. In addition, at 6 weeks post-injury/transplantation, the cells were noted to cross the midline to the contralateral corpus callosum and into the contralateral cortex. Double labeling demonstrated neuronal and astrocytic, but not oligodendrocytic differentiation. Moreover, the cortex appeared to provide an environment that was less hospitable to neuronal differentiation than the hippocampus. CONCLUSIONS: This study shows that expandable human neural stem/progenitor cells survive transplantation, and migrate, differentiate, and proliferate in the injured brain. These cells could potentially be developed for transplantation therapy in cases of traumatic brain injury.

Animals↗

PubMed2003

Neuroprotection by human neural progenitor cells after experimental contusion in rats.

Neural progenitor/stem cells (HNPC) have been suggested to contribute essential trophic factors and promote survival of degenerating neurons after traumatic brain injury. For these reasons we hypothesize that the addition of HNPC to a post-injury region could possibly protect injured neurons. Experimental brain contusions were carried out in 18 rats. Immediately post-injury, rats were injected with 0.1 ml of medium (n=8), dead cells (n=4), or live cells (n=6) in the medial border of the lesion. The rats were sacrificed 6 days post-surgery and evaluated by immunohistochemistry using a human nuclear marker (huN), hematoxylin and Fluoro-Jade (FJ). Human neural stem cells showed engraftment detectable by positive huN staining in 5/6 animals. The non-grafted animal was excluded from further analyses. Those given dead HNPC or medium showed no detectable huN immunoreactivity. A statistical comparison between the numbers of FJ positive degenerating endogenous neurons was made between rats receiving vehicle and dead cells to evaluate whether the presence of human cells would increase neuronal degeneration in comparison to vehicle alone. The rats receiving vehicle had a median of 117.5 FJ positive cells and dead progenitor cell recipients 175.0 per counted section (P<0.05, Mann-Whitney). Consequently, the animals receiving dead human cells were chosen as controls to the animals receiving live progenitor cells. The rats that received live HNPC demonstrated significantly fewer FJ positive cells per counted section than controls (58.0 vs. 175.0, P<0.01, Mann-Whitney). The post-traumatic perilesional environment allowed for the engraftment of live HNPC. The stepwise analysis indicated that host neuronal degeneration was higher in animals transplanted with non-viable human neuronal progenitor cells than in those receiving vehicle, indicating a bystander effect from introducing foreign antigen. In contrast, transplantation of viable progenitor cells attenuated neuronal degeneration, indicating that a potentially beneficial effect in progenitor cell transplantation is not limited to restoration by transplanted cells, but also improving survival of host cells.

Animals↗

PubMed2003

Increases in matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 mRNA after cerebral contusion and depolarisation.

Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play major roles in physiological extracellular matrix turnover during normal development and in pathological processes. In brain, increases in MMP activity occur, for example, in multiple sclerosis, Alzheimer's disease, and after head trauma. We examined MMP-9 and TIMP-1, -2, and -3 in events after head trauma. A time-course study was carried out using two different rat injury models, cerebral contusion and depolarisation. Brains were analysed by RT-PCR and in situ hybridisation. We observed a distinct and time-dependent upregulation of MMP-9 and TIMP-1 mRNA in ipsilateral cortical areas. MMP-9 mRNA levels were upregulated 1 day after cerebral contusion with a peak at Day 4. Depolarisation per se, which also occurs after traumatic brain injury, lead to delayed increase of MMP-9 mRNA, 4 days post application. At Day 14, MMP-9 mRNA levels were indistinguishable from controls in both models. TIMP-1 mRNA increases were observed in both models 4 hr after injury, and increased further at Days 1 and 4. At Day 14, mRNA levels declined and were no higher than control levels. No alterations in mRNA levels were noted for TIMP-2 or -3. Our results support earlier reports on MMP-9 involvement in brain injury. It also shows a role for TIMP-1 in the mechanisms of trauma, where depolarisation could be the mechanism responsible for this upregulation.

Animals↗

PubMed2002

Characterization of Bax and Bcl-2 in apoptosis after experimental traumatic brain injury in the rat.

This study was undertaken to fulfill the need for additional data on the dynamics of Bax and Bcl-2 expression in conjunction to the cell death that ensues following experimental brain contusion. Adult Sprague-Dawley rats were subjected to a unilateral experimental controlled cortical contusion and killed at 1, 2, 4, 6 and 10 days post injury (dpi). Cell death was examined by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method together with immunohistochemistry for cellular markers. Expression of Bax and Bcl-2 were analyzed by immunohistochemistry and in situ hybridization. The number of TUNEL-positive cells was highest at 1 dpi and decreased with time. At all time points, 10-16% of the TUNEL-positive cells showed an apoptotic nuclear morphology. The apoptotic features were restricted to neurons and some inflammatory cells. Immunohistochemistry for Bax revealed a translocation of Bax from a diffuse to a granular distribution in neurons. An up-regulation of Bax mRNA at 6 dpi was discernible. This increase was associated with a statistically significant increase in number of cells with up-regulated and translocated Bax protein. Moreover, a statistically significant increase of Bcl-2 mRNA was detected at 10 dpi. The potential window for anti-apoptotic treatment to salvage neurons is wide. The susceptibility of neurons to necrosis and apoptosis through different pathways during a prolonged post-traumatic period indicate that different pharmacological strategies may be required at different time points after trauma.

Animals↗

PubMed2002

Nitric oxide synthase expression after human brain contusion.

OBJECTIVE: Nitric oxide is a universal mediator of biological effects in the brain, and it has been implicated in the pathophysiological processes of traumatic brain injury. Experimental studies have indicated posttraumatic up-regulation of the three different isoforms of nitric oxide synthase (NOS) (i.e., inducible NOS [iNOS], endothelial NOS, and neuronal NOS) after brain trauma. This study was undertaken to investigate the cellular sources and tissue compartments of nitric oxide produced by human patients undergoing surgical treatment for contusional brain injuries. METHODS: Contused brain tissue specimens from eight consecutive patients who underwent surgical treatment for brain contusions 3 hours to 5 days after trauma were evaluated in immunohistochemical analyses. Double-staining assays were used to define which cells produced the different isoforms. RESULTS: Increases in iNOS-positive cells were detectable within 6 hours after trauma, with a peak at 8 to 23 hours. Expression of iNOS after trauma was detected in neurons, macrophages, neutrophils, astrocytes, and oligodendrocytes. The cellular sources of iNOS differed at different times after trauma. No detectable difference in the expression of the neuronal or endothelial isoforms was observed for trauma patients, compared with control subjects. CONCLUSION: iNOS expression was up-regulated in a time-dependent manner in human brain tissue obtained from patients undergoing surgical treatment for contusional trauma. Our human data largely parallel experimental findings in rats, indicating that such trauma models are relevant for experimental studies and treatment trials.

Adolescent↗

PubMed2000

Possible role of inflammatory mediators in tactile hypersensitivity in rat models of mononeuropathy.

Peripheral hypersensitivity (hyperalgesia and allodynia) are common phenomena both in inflammatory and in neuropathic pain conditions. Several rat models of mononeuropathy (Bennett, Seltzer and Gazelius models) display such symptoms following partial injury to the sciatic nerve. Using immunohistochemistry and behavioral tests, we investigated inflammatory cell and cytokine responses in the sciatic nerve 14 days after injury created in these different models as well as after axotomy. Tactile hypersensitivity ('allodynia') was present in all Gazelius model rats whereas only 38 and 29% of the Bennett and Seltzer models, respectively, displayed this sign of neuropathy. The inflammatory reactions in rats with and without tactile allodynia were compared. Monocytes/macrophages (ED-1), natural killer cells, T lymphocytes, and the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), were significantly upregulated in all nerve injured rats in comparison to sham-operated controls. Interestingly, ED-1-, TNF-alpha- and IL-6-positive cells increased more markedly in allodynic Bennett and Seltzer rats than in non-allodynic ones. The magnitude of the inflammatory response does not seem to relate to the extent of damage to the nerve fibers because axotomized rats displayed much lower upregulation. Our findings indicate that the considerable increase in monocytes/macrophages induced by a nerve injury results in a very high release of IL-6 and TNF-alpha. This may relate to the generation of tactile allodynia/hyperalgesia, since there was a clear correlation between the number of ED-1 and IL-6-positive cells and the degree of allodynia. It is possible that measures to reduce monocyte/macrophage recruitment and the release of pro-inflammatory interleukins after nerve damage could influence the development of neuropathic pain.

Animals↗