Apoptotic cell death progression after photothrombotic focal cerebral ischaemia: effects of the lipophilic iron chelator 2,2'-dipyridyl

Two different forms of cell death have been distinguished morphologically following cerebral ischaemia: necrotic and apoptotic cell death. The aim of this study was to investigate the contribution of apoptosis to ischaemic damage by carefully depicting the temporal and spatial neuronal death following focal ischaemia. For this purpose, rats were subjected to chemical photothrombosis, and histological and biochemical analyses were performed over a period of 24 h after the onset of ischaemia. In addition, the effects of the lipophilic antioxidant iron chelator 2,2'-dipyridyl (DP) were evaluated 24 h after photothrombosis when the lesion volume was maximal. Our results showed two separate waves of neuronal death. In the first wave, shrunken dark neurons were massively present as early as 2 h after photothrombosis in the infarct core. From this initial neuronal abnormal population, progressive and time-dependent changes of both necrotic and apoptotic cell death were observed, leading to ghost neurons and apoptotic bodies after 24 h. The extension of the lesion coincided with a second wave of cell death. Massive and rapid neuronal loss occurred at the infarct border, which appeared as a sharply demarcated pale region. Procaspase and poly(ADP-ribose) polymerase-1 (PARP-1) cleavages were also detected in the infarct core and surrounding damaged tissue. DP treatment markedly blocked the enlargement of the lesion, the infarct border being rescued from infarction. Furthermore, a large decrease of apoptotic bodies was associated with a significant drop of caspase and PARP-1 cleavages, suggesting that the protective effect of DP closely correlates with limitation of apoptosis expansion.     

Involvement of thrombin and mitogen-activated protein kinase pathways in hemorrhagic brain injury

Thrombin is thought to play an important role in brain damage associated with intracerebral hemorrhage (ICH). We previously showed that activation of mitogen-activated protein (MAP) kinases and recruitment of microglia are crucial for thrombin-induced shrinkage of the striatal tissue in vitro and thrombin-induced striatal damage in vivo. Here we investigated whether the same mechanisms are involved in ICH-induced brain injury. A substantial loss of neurons was observed in the center and the peripheral region of hematoma at 3 days after ICH induced by intrastriatal injection of collagenase in adult rats. Intracerebroventricular injection of argatroban or cycloheximide, both of which prevent thrombin cytotoxicity in vitro, exhibited a significant neuroprotective effect against ICH-induced injury. ICH-induced neuron loss was also prevented by a MAP kinase kinase inhibitor (PD98059) and a c-Jun N-terminal kinase inhibitor (SP600125). These drugs had no effect on hematoma size or ICH-induced brain edema. Activation of extracellular signal-regulated kinase in response to ICH was observed in both neurons and microglia. Despite their neuroprotective effects, MAP kinase inhibitors did not decrease the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appearing after ICH. Identification of cell types revealed that TUNEL staining occurred prominently in neurons but not in microglia, whereas inhibition of MAP kinases resulted in appearance of TUNEL staining in microglia. These results suggest that thrombin and the activation of MAP kinases are involved in ICH-induced neuronal injury, and that neuroprotective effects of MAP kinases are in part mediated by arrestment of microglial activities.     

Noninvasive magnetic resonance imaging stratifies injury severity in a rodent model of male juvenile traumatic brain injury

Age and severity are significant predictors of traumatic brain injury (TBI) outcomes in the immature brain. TBI studies have segregated TBI injury into three severity groups: mild, moderate, and severe. While mild TBI is most frequent form in children and adults, there is debate over the indicators used to denote mild injury. Clinically, magnetic resonance imaging (MRI) and computed tomography (CT) are used to diagnose the TBI severity when medically warranted. Herein, we induced mild, moderate, and severe TBI in juvenile rats (jTBI) using the controlled cortical impact model. We characterized the temporal and spatial injury after graded jTBI in vivo using high-field MRI at 0.25 (6 hr), 1 and 3 days post-injury (dpi) with comparative histology. Susceptibility-weighted imaging (SWI) for blood and T2-weighted imaging (T2WI) for edema were quantified over the 0.25–3 dpi. Edema volumes increased linearly with severity at 0.25 dpi that slowly continued to decrease over the 3 dpi. In contrast, blood volumes did not decrease over time. Mild TBI had the least amount of blood visible on SWI. Fluoro-jade B (FJB) staining for cell death confirmed increased cellular death with increasing severity and increased FJB + cells in the corpus callosum (CC). Interestingly, the strongest correlation was observed for cell death and the presence of extravascular blood. A clear understanding of acute brain injury (jTBI) and how blood/edema contribute to mild, moderate, and severe jTBI is needed prior to embarking on therapeutic interventions. Noninvasive imaging should be used in mild jTBI to verify lack of overt injury.     

脑出血后脑水含量变化及铁络合剂的作用

目的探讨脑出血后脑水肿的发生机制及铁络合剂的作用。方法将0．3ml自体血和0．3ml自体血 100mg去铁胺分别注入兔的两侧额叶,测定血肿周围脑组织的水含量。结果自体血注射后4h开始出现水肿,72h达高峰;去铁胺可减轻其水肿程度。结论红细胞在脑出血后脑水肿的形成中起重要作用;铁络合剂可减轻脑出血后脑水肿。     

FK506 ameliorates the discrimination learning impairment due to preventing the rarefaction of white matter induced by chronic cerebral hypoperfusion in rats

We examined the effects of the immunosuppressant tacrolimus (FK506) on the discrimination learning impairment induced by chronic cerebral hypoperfusion in rats. Chronic cerebral hypoperfusion was prepared by permanent ligation of bilateral common carotid arteries for male Wistar rats aged 9 weeks. FK506 (0.05 mg/kg, s.c.) recovered the learning impairment and also prevented the rarefaction of white matter and striatal neuronal cell damage. Our findings suggest that FK506 ameliorates the learning impairment mainly due to preventing neuropathological alterations.     

Neurons lacking iron regulatory protein-2 are highly resistant to the toxicity of hemoglobin

The effect of iron regulatory protein-2 (IRP2) on ferritin expression and neuronal vulnerability to hemoglobin was assessed in primary cortical cell cultures prepared from wild-type and IRP2 knockout mice. Baseline levels of H and L-ferritin subunits were significantly increased in IRP2 knockout neurons and astrocytes. Hemoglobin was toxic to wild-type neurons in mixed neuron–astrocyte cultures, with an LC₅₀ near 3 µM for a 24 h exposure. Neuronal death was reduced by 85–95% in knockout cultures, and also in cultures containing knockout neurons plated on wild-type astrocytes. Protein carbonylation, reactive oxygen species formation, and heme oxygenase-1 expression after hemoglobin treatment were also attenuated by IRP2 gene deletion. These results suggest that IRP2 binding activity increases the vulnerability of neurons to hemoglobin, possibly by reducing ferritin expression. Therapeutic strategies that target this regulatory mechanism may be beneficial after hemorrhagic CNS injuries.     

The density and distribution of ischemic brain injury in the rat following 2–10 min of forebrain ischemia

Summary The density and distribution of brain damage after 2–10 min of cerebral ischemia was studied in the rat. Ischemia was produced by a combination of carotid clamping and hypotension, followed by 1 week recovery. The brains were perfusion-fixed with formaldehyde, embedded in paraffin, subserially sectioned, and stained with acid fuchsin/cresyl violet. The number of necrotic neurons in the cerebral cortex, hippocampus, and caudate nucleus was assessed by direct visual counting.Somewhat unexpectedly, mild brain damage was observed in some animals already after 2 min, and more consistently after 4 min of ischemia. This damage affected CA4 and CA1 pyramids in the hippocampus, and neurons in the subiculum. Necrosis of neocortical cells began to appear after 4 min and CA3 hippocampal damage after 6 min of ischemia, while neurons in the caudoputamen were affected first after 8–10 min.Selective neuronal necrosis of the cerebral cortex worsened into infarction after higher doses of insult. Damage was worst over the superolateral convexity of the hemisphere, in the middle laminae of the cerebral cortex. The caudate nucleus showed geographically demarcated zones of selective neuronal necrosis, damage to neurons in the dorsolateral portion showing an all-or-none pattern. Other structures involved included the amygdaloid, the thalamic reticular nucleus, the septal nuclei, the pars reticularis of the substantia nigra, and the cerebellar vermis.Supported by the Swedish Medical Research Council (projects 12X-03020, 14X-263) and the National Institutes of Health of the United States Public Health Service (grant no. 5 R01 NS07838). Dr. Auer is the recipient of a Medical Research Council of Canada Fellowship.     

Regional and temporal characterization of neuronal, glial, and axonal response after traumatic brain injury in the mouse

Fluid percussion injury (FPI) is a commonly used and clinically relevant model of traumatic brain injury (TBI) in the rat. Recently, our lab successfully adapted FPI to mice. To account for differences in response to injury between mice and rats and provide a foundation for further use of FPI in gene-targeting studies, we sought to characterize the temporal and regional response to FPI in male C57BL/6 mice. Animals were killed at 10 min, 24 h, and 4, 7, 14, and 35 days (n = 3 for each group) after a very severe parasagittal FPI (> 4.0 atm) or sham injury (n = 3). Extensive numbers of damaged neurons were consistently found in the ipsilateral cortex, thalamus, and hippocampus by 10 min. This damage was nearly identical at 24 h, but quickly declined at subsequent time points. Activated microglia were found only in regions of neuronal injury at the earliest time points. Glial fibrillary acidic protein immunoreactivity reached significantly higher levels compared with controls at 7 days (P < 0.05) in the cortex, thalamus, and hippocampus and remained elevated for 35 days. White matter degeneration was present in all regions examined. This damage did not appear until at least day 4, but progressed up to day 35. The spatial pattern of damage we observed in mice after FPI is similar to that seen in rats. However, the temporal progression of neuronal injury in mice is comparatively abbreviated in the hippocampus and thalamus. In conclusion, these results suggest that FPI in mice may be a particularly useful tool for studying mechanisms of TBI in gene-targeting studies. Received: 13 October 1998 / Revised: 22 February 1999 / Accepted: 24 March 1999     

Hemoglobin and iron handling in brain after subarachnoid hemorrhage and the effect of deferoxamine on early brain injury

The purpose of this study was to investigate hemoglobin and iron handling after subarachnoid hemorrhage (SAH), examine the relationship between iron and neuroglial cell changes, and determine whether deferoxamine (DFX) can reduce SAH-induced injury. The SAH was induced in Sprague-Dawley rats (n=110) using an endovascular perforation technique. Animals were treated with DFX (100 mg/kg) or vehicle 2 and 6 hours after SAH induction followed by every 12 hours for 3 days. Rats were killed at 6 hours, Days 1 and 3 to determine nonheme iron and examine iron-handling proteins using Western blot and immunohistochemistry. 8-Hydroxyl-2′-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining were performed to assess oxidative DNA damage and neuronal cell death. After SAH, marked heme-oxygenase-1 (HO-1) upregulation at Day 3 (P<0.01) was accompanied by elevated nonheme iron (P<0.01), transferrin (Tf) (P<0.01), Tf receptor (P<0.05), and ferritin levels (P<0.01). Deferoxamine treatment reduced SAH-induced mortality (12% versus 29%, P<0.05), brain nonheme iron concentration, iron-handling protein expression, oxidative stress, and neuronal cell death at Day 3 (P<0.01) after SAH. These results suggest that iron overload in the acute phase of SAH causes oxidative injury leading to neuronal cell death. Deferoxamine effectively reduced oxidative stress and neuronal cell death, and may be a potential therapeutic agent for SAH.     

Light and electron microscopic assessment of progressive atrophy following moderate traumatic brain injury in the rat

The presence of progressive white matter atrophy following traumatic brain injury (TBI) has been reported in humans as well as in animal models. However, a quantitative analysis of progressive alterations in myelinated axons and other cellular responses to trauma has not been conducted. This study examined quantitative differences in myelinated axons from several white and gray matter structures between non-traumatized and traumatized areas at several time points up to 1 year. We hypothesize that axonal numbers decrease over time within the structures analyzed, based on our previous work demonstrating shrinkage of tissue in these vulnerable areas. Intubated, anesthetized male Sprague-Dawley rats were subjected to moderate (1.8–2.2 atm) parasagittal fluid-percussion brain injury, and perfused at various intervals after surgery. Sections from the fimbria, external capsule, thalamus and cerebral cortex from the ipsilateral hemisphere of traumatized and sham-operated animals were prepared and. estimated total numbers of myelinated axons were determined by systematic random sampling. Electron micrographs were obtained for ultrastructural analysis. A significant (P<0.05) reduction in the number of myelinated axons in the traumatized hemisphere compared to control in all structures was observed. In addition, thalamic and cortical axonal counts decreased significantly (P<0.05) over time. Swollen axons and macrophage/microglia infiltration were present as late as 6 months post-TBI in various structures. This study is the first to describe quantitatively chronic axonal changes in vulnerable brains regions after injury. Based on these data, a time-dependent decrease in the number of myelinated axons is seen to occur in vulnerable gray matter regions including the cerebral cortex and thalamus along with distinct morphological changes within white matter tracts after TBI. Although this progressive axonal response to TBI may include Wallerian degeneration, other potential mechanisms underlying this progressive pathological response within the white matter are discussed.     

Dose-dependent, protective effect of FK506 against white matter changes in the rat brain after chronic cerebral ischemia

Neuroprotective effects of immunosuppressive agents have been shown in cerebral ischemia. To investigate the role of immunosuppressive agents in chronic cerebral ischemia and to design a drug protocol with safe therapeutic windows, we examined the effects of FK506, a potent immunosuppressive agent, on chronic cerebral ischemia. Both common carotid arteries were ligated in 73 male Wistar rats. Fifty-eight of these rats received a chronic injection of FK506 (0.2, 0.5, 1.0 mg/kg) and the remaining 15 received a vehicle solution injection. Microglia/macrophage was investigated with immunohistochemistry for leukocyte common antigen and major histocompatibility complex, and astroglia was examined with glial fibrillary acidic protein as markers. White matter rarefaction and the number of immunopositive glial cells were assessed from 7 to 30 days after the ligation. In the vehicle-treated animals, there was persistent and extensive activation of the microglia/macrophages and astroglia in the white matter, including the optic nerve, optic tract, corpus callosum, internal capsule, anterior commissure and traversing fiber bundles of the caudoputamen. In the FK506-treated rats, the number of activated microglia/macrophages was significantly reduced in a dose-dependent manner (p<0.01) as compared to the vehicle-treated rats. Rarefaction of the white matter was also inhibited by FK506 in a dose-dependent manner (p<0.01). Thus, a clinically-relevant dosage of FK506 attenuated both glial activation and white matter changes in chronic cerebral ischemia in the rat. These results indicate a potential use for FK506 in cerebrovascular diseases.     

Efficacy and safety of deferiprone for the treatment of pantothenate kinase-associated neurodegeneration (PKAN) and neurodegeneration with brain iron accumulation (NBIA): Results from a four years follow-up

ObjectiveTo evaluate the long-term effect of Deferiprone (DFP) in reducing brain iron overload and improving neurological manifestations in patients with NBIA.Methods6 NBIA patients (5 with genetically confirmed PKAN), received DFP solution at 15 mg/kg po bid. They were assessed by UPDRS/III and UDRS scales and blinded video rating, performed at baseline and every six months. All patients underwent brain MRI at baseline and during follow up. Quantitative assessment of brain iron was performed with T2* relaxometry, using a gradient multi-echo T2* sequence.ResultsAfter 48 months of treatment clinical rating scales and blinded video rating indicated a stabilization in motor symptoms in 5/6 Pts. In the same subjects MRI evaluation showed reduced hypointensity in the globus pallidus (GP); quantitative assessment confirmed a significant increment in the T2* value, and hence reduction of the iron content of the GP.ConclusionThe data from our 4-years follow-up study confirm the safety of DFP as a chelator agent for iron accumulation. The clinical stabilization observed in 5/6 of our patients suggests that DFP may be a reasonable therapeutic option for the treatment of the neurological manifestations linked with iron accumulation and neurodegeneration, especially in adult patients at early stage of the disease.(Clinicaltrials.gov identifier: NTC00907283).     

Improvement of brain energy metabolism and cholinergic functions contributes to the beneficial effects of silibinin against streptozotocin induced memory impairment

Recently, silibinin, a clinically used hepatoprotectant, has been reported to prevent amyloid beta induced memory impairment by reducing oxidative stress and inflammation in mice brain. However, the exact mechanism of neuroprotective effect of silibinin has not been properly studied especially in context of brain energy metabolism and cholinergic functions, the essential factors that undergo impairment in Alzheimer's disease. Therefore, the present study investigated the effect of silibinin on impairment in memory, brain energy metabolism and cholinergic function following intracerebral (IC) streptozotocin (STZ) administration in mice. STZ (0.5 mg/kg), administered twice at an interval of 48 h, caused significant memory impairment tested by Morris water maze. Further, STZ significantly decreased ATP and increased synaptosomal calcium level in mice brain. Increased oxidative and nitrosative stress was also observed in IC STZ injected mice brain. STZ IC induced memory impairment is associated with increased activity and mRNA expression of acetylcholinesterase (AChE) and decreased α-7 nicotinic acetylcholine receptor (α-7-nAChR) mRNA expression in mice brain. Pretreatment with silibinin (100 and 200 mg/kg, po) attenuated STZ induced memory impairment by reducing oxidative and nitrosative stress and synaptosomal calcium ion level. Further, silibinin dose dependently restored ATP level indicating improvement in brain energy metabolism. The activity and mRNA expression of AChE was restored by silibinin. Moreover, α-7-nAChR mRNA expression was significantly increased by silibinin in STZ treated mice brain. The present study clearly demonstrates that beneficial effects of silibinin in STZ induced memory impairment in mice is due to improvement in brain energy metabolism and cholinergic function.