Xiurong Zhao

Molecular Pathophysiology of Cerebral Hemorrhage Secondary Brain Injury

Intracerebral hemorrhage (ICH) is an often fatal type of stroke that kills approximately 30,000 people annually in the United States. If the patient survives the ictus, then the resulting hematoma within brain parenchyma triggers a series of adverse events causing secondary insults and severe neurological deficits. This article discusses selected aspects of secondary brain injury after ICH and outlines key mechanisms associated with hematoma toxicity, oxidative stress, and inflammation. Finally, this review discusses the relevance of hematoma resolution processes as a target for ICH therapy and presents potential clinically relevant molecular targets that could be harnessed to treat secondary injury associated with ICH injury.

15d-Prostaglandin J2 Activates Peroxisome Proliferator-Activated Receptor-γ, Promotes Expression of Catalase, and Reduces Inflammation, Behavioral Dysfunction, and Neuronal Loss after Intracerebral Hemorrhage in Rats:

Peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that regulates the expression of various gene products that are essential in lipid and glucose metabolism, as well as that of the peroxisome-enriched antioxidant enzyme, catalase. Activation of PPARγ is linked to anti-inflammatory activities and is beneficial for cardiovascular diseases. However, little is known about its role in intracerebral hemorrhage (ICH). 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) acts as a physiologic agonist for PPARγ. In this study, we found that injection of 15d-PGJ2 into the locus of striatal hematoma increased PPARγ-deoxyribonucleic acid (DNA) binding activity and the expression of catalase messenger ribonucleic acid (mRNA) and protein in the perihemorrhagic area. Additionally, 15d-PGJ2 significantly reduced nuclear factor-κB (NF-κB) activation and prevented neutrophil infiltration measured by myeloperoxidase (MPO) immunoassay, and also reduced cell apoptosis measured by terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL). In addition, 15d-PGJ2 reduced behavioral dysfunction produced by the ICH. Altogether, our findings indicate that injection of 15d-PGJ2 at the onset of ICH is associated with activation of PPARγ and elevation of catalase expression, suppression of NF-κB activity, and restricted neutrophil infiltration. All these events predicted reduced behavioral deficit and neuronal damage.

Regional calpain and caspase-3 proteolysis of α-spectrin after traumatic brain injury

ACTIVITY of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein α-spectrin into signature spectrin breakdown products (SBDPs) was used to provide the first systematic and simultaneous comparison of changes in activity of these two families of cysteine proteases after traumatic brain injury (TBI) in rats. Distinct regional and temporal patterns of calpain/caspase-3 processing of α-spectrin were observed in brain regions ipsilateral to the site of injury after TBI, including large increases of 145 kDa calpain-mediated SBDP in cortex (up to 30-fold), and enduring increases (up to 2 weeks) of 145 kDa SBDP in hippocampus and thalamus. By contrast, 120 kDa caspase-3-mediated SBDP was absent in cortex and showed up to a 2-fold increase in hippocampus and striatum at early (hours) after TBI. Future studies will clarify the pathological significance of large regional differences in activation of calpain and caspase-3 proteases after TBI.

Hematoma resolution as a target for intracerebral hemorrhage treatment: Role for peroxisome proliferator‐activated receptor γ in microglia/macrophages

Phagocytosis is necessary to eliminate the hematoma after intracerebral hemorrhage (ICH); however, release of proinflammatory mediators and free radicals during phagocyte activation is toxic to neighboring cells, leading to secondary brain injury. Promotion of phagocytosis in a timely and efficient manner may limit the toxic effects of persistent blood products on surrounding tissue and may be important for recovery after ICH.

A calpain inhibitor attenuates cortical cytoskeletal protein loss after experimental traumatic brain injury in the rat

The capacity of a calpain inhibitor to reduce losses of neurofilament 200-, neurofilament 68- and calpain 1-mediated spectrin breakdown products was examined following traumatic brain injury in the rat. Twenty-four hours after unilateral cortical impact injury, western blot analyses detected neurofilament 200 losses of 65% (ipsilateral) and 36% (contralateral) of levels observed in naive, uninjured rat cortices. Neurofilament 68 protein levels decreased only in the ipsilateral cortex by 35% relative to naive protein levels. Calpain inhibitor 2, administered 10 min after injury via continuous arterial infusion into the right external carotid artery for 24 h, significantly reduced neurofilament 200 losses to 17% and 3% relative to naive neurofilament 200 protein levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor administration abolished neurofilament 68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain-mediated neurofilament 68 breakdown products. Spectrin breakdown products mediated by calpain 1 activation were detectable in both hemispheres 24 h after traumatic brain injury and were substantially reduced in animals treated with calpain inhibitor 2 both ipsilaterally and contralaterally to the site of injury. Qualitative immunofluorescence studies of neurofilament 200 and neurofilament 68 confirmed western blot data, demonstrating morphological protection of neuronal structure throughout cortical regions of the traumatically injured brain. Morphological protection included preservation of dendritic structure and reduction of axonal retraction balls. In addition, histopathological studies employing hematoxylin and eosin staining indicated reduced extent of contusion at the injury site. These data indicate that calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons after experimental traumatic brain injury in vivo.

Temporal Profile of Apoptotic-like Changes in Neurons and Astrocytes Following Controlled Cortical Impact Injury in the Rat

Apoptotic cell death has been observed in both neurodegenerative diseases and acute neurological traumas such as ischemia, spinal cord injury, and traumatic brain injury (TBI). Recent studies employing different models of TBI have described morphological and biochemical changes characteristic of apoptosis following injury. However, no study has examined the temporal profile of apoptosis following controlled cortical impact (CCI) injury in the rat. In addition, the relative frequency of apoptotic profiles in different cell types (neurons versus glia) following CCI has yet to be investigated. In the present experiments, injured cortex was subjected to DNA electrophoresis, and serial sections from the contusion area were stained with hematoxylin and eosin or Hoechst 33258 or double-labeled with TUNEL and neuronal or glial markers. The results of the present study indicate that CCI produces a substantial amount of DNA damage associated with both apoptotic-like and necrotic-like cell death phenotypes primarily at the site of cortical impact and focal contusion. DNA damage, as measured by TUNEL and DNA electrophoresis, was most apparent 1 day following injury and absent by 14 days post-TBI. However, quantitative analysis showed that the majority of TUNEL-positive cells failed to exhibit apoptotic-like morphology and were probably undergoing necrosis. In addition, apoptotic-like morphology was predominantly observed in neurons compared to astrocytes. The present study provides further evidence that apoptosis is involved in the pathology of TBI and could contribute to some of the ensuing cell death following injury.

Transcription Factor Nrf2 Protects the Brain From Damage Produced by Intracerebral Hemorrhage

Background and Purpose— Intracerebral hemorrhage (ICH) remains a major medical problem for which there is no effective treatment. Oxidative and cytotoxic damage plays an important role in ICH pathogenesis and may represent a target for treatment of ICH. Recent studies have suggested that nuclear factor–erythroid 2–related factor 2 (Nrf2), a pleiotropic transcription factor, may play a key role in protecting cells from cytotoxic/oxidative damage. This study evaluated the role of Nrf2 in protecting the brain from ICH-mediated damage. Methods— Sprague-Dawley rats and Nrf2-deficient or control mice received intracerebral injection of autologous blood to mimic ICH. Sulforaphane was used to activate Nrf2. Oxidative stress, the presence of myeloperoxidase-positive cells (neutrophils) in ICH-affected brains, and behavioral dysfunction were assessed to determine the extent of ICH-mediated damage. Results— Sulforaphane activated Nrf2 in ICH-affected brain tissue and reduced neutrophil count, oxidative damage, and behavioral deficits caused by ICH. Nrf2-deficient mice demonstrated more severe neurologic deficits after ICH and did not benefit from the protective effect of sulforaphane. Conclusions— Nrf2 may represent a strategic target for ICH therapies.

μ-Calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury

Abstract: Increasing evidence suggests that excessive activation of the calcium‐activated neutral protease μ‐calpain could play a major role in calcium‐mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ‐calpain after unilateral cortical impact injury in vivo, the ratio of the 76‐kDa activated isoform of μ‐calpain to its 80‐kDa precursor was measured by western blotting. This μ‐calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ‐calpain activation on proteolysis of the neuronal cytoskeletal protein α‐spectrin. Immunoreactivity for α‐spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α‐spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α‐spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ‐calpain autolysis, calpain‐mediated α‐spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ‐calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain‐mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ‐calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Temporal Profile and Cell Subtype Distribution of Activated Caspase‐3 Following Experimental Traumatic Brain Injury

Abstract: This study investigated the temporal expression and cell subtype distribution of activated caspase‐3 following cortical impact‐induced traumatic brain injury in rats. The animals were killed and examined for protein expression of the proteolytically active subunit of caspase‐3, p18, at intervals from 6 h to 14 days after injury. In addition, we also investigated the effect of caspase‐3 activation on proteolysis of the cytoskeletal protein α‐spectrin. Increased protein levels of p18 and the caspase‐3‐specific 120‐kDa breakdown product to α‐spectrin were seen in the cortex ipsilateral to the injury site from 6 to 72 h after the trauma. Immunohistological examinations revealed increased expression of p18 in neurons, astrocytes, and oligodendrocytes from 6 to 72 h following impact injury. In contrast, no evidence of caspase‐3 activation was seen in microglia at all time points investigated. Quantitative analysis of caspase‐3‐positive cells revealed that the number of caspase‐3‐positive neurons exceeded the number of caspase‐3‐positive glia cells from 6 to 72 h after injury. Moreover, concurrent assessment of nuclear histopathology using hematoxylin identified p18‐immunopositive cells exhibiting apoptotic‐like morphological profiles in the cortex ipsilateral to the injury site. In contrast, no evidence of increased p18 expression or α‐spectrin proteolysis was seen in the ipsilateral hippocampus, contralateral cortex, or hippocampus up to 14 days after the impact. Our results are the first to demonstrate the concurrent expression of activated caspase‐3 in different CNS cells after traumatic brain injury in the rat. Our findings also suggest a contributory role of activated caspase‐3 in neuronal and glial apoptotic degeneration after experimental TBI in vivo.

Altered PPARγ expression and activation after transient focal ischemia in rats

Stroke is a devastating disease with limited treatment options. Recently, we found that the peroxisome proliferator‐activated receptor‐γ (PPARγ) agonists troglitazone and pioglitazone reduce injury and inflammation in a rat model of transient cerebral ischemia. The mechanism of this protection is unclear, as these agents can act through PPAR‐γ activation or through PPAR‐γ‐independent mechanisms. Therefore, we examined PPAR‐γ expression, DNA binding and transcriptional activity following stroke. In addition, we used a PPAR‐γ antagonist, T0070907, to determine the role of PPAR‐γ during ischemia. Using immunohistochemical techniques and real‐time PCR, we found low levels of PPAR‐γ mRNA and PPAR‐γ immunoreactivity in nonischemic brain; however, PPAR‐γ expression dramatically increased in ischemic neurons, peaking 24u2003h following middle cerebral artery occlusion. Interestingly, we found that in both vehicle‐ and agonist‐treated brains, DNA binding was reduced in the ischemic hemisphere relative to the contralateral hemisphere. Expression of a PPAR‐γ target gene, lipoprotein lipase, was also reduced in ischemic relative to nonischemic brain. Both DNA binding and lipoprotein lipase expression were increased by the addition of the PPAR‐γ agonist rosiglitazone. Finally, we found that rosiglitazone‐mediated protection after stroke was reversed by the PPAR‐γ antagonist T0070907. Interestingly, infarction size was also increased by T0070907 in the absence of PPAR‐γ agonist, suggesting that endogenous PPAR‐γ ligands may mitigate the effects of cerebral ischemia.