Elsevier

Brain Research

Volume 1249, 16 January 2009, Pages 229-236
Brain Research

Research Report
Tissue-type transglutaminase and the effects of cystamine on intracerebral hemorrhage-induced brain edema and neurological deficits

https://doi.org/10.1016/j.brainres.2008.10.035Get rights and content

Abstract

Neurodegeneration occurs after intracerebral hemorrhage (ICH) and tissue-type transglutaminase (tTG) has a role in neurodegenerative disorders. The present study investigated tTG expression after ICH and the effects of a tTG inhibitor, cystamine, on ICH-induced brain edema and neurological deficits. This study has two parts. In the first, male Sprague-Dawley rats received an intracaudate injection of 100 µL autologous whole blood or a needle insertion (sham). Rats were killed 3 days later and the brains used for immunohistochemistry, Western blots and real-time quantitative polymerase chain reaction. In the second set, ICH rats were treated intraperitoneally with either a tTG inhibitor, cystamine, or vehicle. Rats underwent behavioral testing and were killed at day-3 for measurement of brain swelling. tTG positive cells were found in the ipsilateral basal ganglia after ICH and most of those cells were neuron-like. Western blot analysis showed a 3-fold increase in tTG in the ipsilateral basal ganglia (p < 0.01 vs. sham) after ICH. tTG mRNA levels were also significantly higher (8.5-fold increase vs. sham). Cystamine treatment attenuated ICH-induced brain swelling (day 3: 14.4 ± 3.2 vs. 21.4 ± 4.0% in vehicle-treated rats, p < 0.01), neuronal death and improved functional outcome (forelimb placing score: 47 ± 23 vs. 17 ± 16% in vehicle-treated rats, p < 0.05). ICH induces perihematomal tTG upregulation and cystamine, a tTG inhibitor, reduces ICH-induced brain swelling and neurological deficits.

Introduction

Spontaneous intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype. Community based studies have indicated a mortality of more than 40%, and many survivors are left with significant neurological deficits (Mendelow et al., 2005). The appropriate therapy for ICH remains a subject of debate, and an improved understanding of the detailed cellular and biochemical mechanisms leading to neuronal injury after ICH may facilitate the development of improved medical and surgical therapies. Brain edema aggravates brain injury and is associated with poor outcome in patients (Zazulia et al., 1999). There are several phases of edema formation after ICH including coagulation cascade, thrombin production, erythrocyte lysis, and hemoglobin toxicity (Xi et al., 2006). Further, a relationship between ICH and neuronal cell death has been noted (Kingman et al., 1988).

Transglutaminases are a family of cross-linking enzymes catalyzing the formation of γ-glutamyl-ε-lysine bonds. Tissue-type transglutaminase (tTG) is the most ubiquitously expressed member of this family. It is abundantly expressed in the brain (Kim et al., 1999) and an increase in tTG mRNA levels has been observed during normal aging in humans (Lu et al., 2004). Expression of tTG is implicated in numerous processes including neurodegeneration (Citron et al., 2002; and Lesort et al., 2000). An upregulation in tTG has been documented in chronic neuropathological conditions such as Huntington disease, Parkinson's disease, and Alzheimer's disease (Citron et al., 2002; and Lesort et al., 2000). Because tTG upregulation during cell death processes is well documented in many different models (Piacentini et al., 2005), it has been hypothesized that tTG can facilitate neuronal cell death in neurodegenerative disease as well as in CNS injury. However, the role of tTG in ICH-induced brain injury has not yet been examined.

Cystamine is a competitive inhibitor of TG activity that has been shown to limit the aggregation of proteins with expanded polyglutamine tracts in vitro (Igarashi et al., 1998). Several studies have demonstrated that cystamine treatment is neuroprotective in Huntington disease (Karpuj et al., 2002, Van Raamsdonk et al., 2005; and Wang et al., 2005). Furthermore, it has been described that cystamine can also inhibit caspase-3 activity (Lesort et al., 2003), increase intracellular levels of the antioxidants glutathione (Lesort et al., 2003), and increase the expression of heat-shock proteins (Karpuj et al., 2002).

In this study, we examined brain protein and mRNA levels of tTG in a rat model of ICH. We also investigated the effects of the tTG inhibitor, cystamine, on brain edema and functional outcomes following ICH.

Section snippets

Physiological variables

All physiological variables were measured immediately before an ICH. Mean arterial blood pressure, blood pH, PaO2, PaCO2, and blood glucose level were controlled within normal ranges (data not shown).

Brain tTG levels after ICH

Immunohistochemistry demonstrated that tTG protein was over-expressed in the ipsilateral basal ganglia after ICH (Fig. 1Ab) compared with the contralateral basal ganglia (Fig. 1Ac) or the ipsilateral basal ganglia after needle insertion (Fig. 1Aa). Immuno-fluorescent double labeling showed that

Discussion

This study demonstrates that brain tTG mRNA and protein levels are increased in the perihematomal area after ICH. Previous studies have shown tTG upregulation in animal models of cerebral ischemia (Ientile et al., 2004; and Tolentino et al., 2004), traumatic brain injury (Tolentino et al., 2002), calcium-induced hippocampal damage (Tucholski et al., 2006), and spinal cord injury (Festoff et al., 2002). Evidence is mounting that tTG may have a role in acute brain injury (as explored here for

Animal preparation and intracerebral infusion

Animal use protocols were approved by the University of Michigan Committee on the Use and Care of Animals. A total of thirty male Sprague-Dawley rats (weight, 300 to 350 g; Charles River Laboratories, Portage, MI, U.S.A.) were used in this study. Rats were anesthetized with intraperitoneal pentobarbital (45 mg/kg). The right femoral artery was catheterized for continuous blood pressure monitoring and blood sampling. Blood was obtained from the catheter for analysis of blood pH, PaO2, PaCO2, and

Acknowledgments

This study was supported by grants NS-017760, NS-039866, NS-047245 and NS-057539 from the National Institutes of Health (NIH) and 0435354Z and 0840016N from the American Heart Association (AHA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH and AHA.

References (44)

  • WangX. et al.

    Cerebral PET imaging and histological evidence of transglutaminase inhibitor cystamine induced neuroprotection in transgenic R6/2 mouse model of Huntington's disease

    J. Neurol. Sci.

    (2005)
  • WuJ. et al.

    Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage

    Brain Res.

    (2002)
  • XiG. et al.

    Mechanisms of brain injury after intracerebral haemorrhage

    Lancet Neurol.

    (2006)
  • Borrell-PagesM. et al.

    Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase

    J. Clin. Invest.

    (2006)
  • FestoffB.W. et al.

    Injury-induced “switch” from GTP-regulated to novel GTP-independent isoform of tissue transglutaminase in the rat spinal cord

    J. Neurochem.

    (2002)
  • FesusL.

    Transglutaminase-catalyzed protein cross-linking in the molecular program of apoptosis and its relationship to neuronal processes

    Cell. Mol. Neurobiol.

    (1998)
  • FujitaK. et al.

    Protective effect against 17beta-estradiol on neuronal apoptosis in hippocampus tissue following transient ischemia/recirculation in Mongolian gerbils via down-regulation of tissue transglutaminase activity

    Neurochem. Res.

    (2006)
  • GongC. et al.

    Intracerebral hemorrhage-induced neuronal death

    Neurosurgery

    (2001)
  • HuaY. et al.

    Behavioral tests after intracerebral hemorrhage in the rat

    Stroke

    (2002)
  • HuangF. et al.

    Brain edema after experimental intracerebral hemorrhage: role of hemoglobin degradation products

    J. Neurosurg.

    (2002)
  • IentileR. et al.

    Cystamine inhibits transglutaminase and caspase-3 cleavage in glutamate-exposed astroglial cells

    J. Neurosci. Res.

    (2003)
  • IgarashiS. et al.

    Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch

    Nat. Genet.

    (1998)
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