Combination of Thrombin and Matrix Metalloproteinase-9 Exacerbates Neurotoxicity in Cell Culture and Intracerebral Hemorrhage in Mice

Abstract

The rapid loss of neurons is a major pathological outcome of intracerebral hemorrhage (ICH). Several mechanisms may produce the neurotoxicity observed in ICH, and these include proteolytic enzymes such as thrombin and matrix metalloproteinase-9 (MMP-9). We tested the hypothesis that thrombin and MMP-9 combine to injure neurons in culture and that they interact to promote the acute neurotoxicity that occurs in ICH in vivo. We report that human fetal neurons die when exposed to thrombin or MMP-9 in isolation and that a combination of these two enzymes increased neurotoxicity. The toxicity of thrombin involved protease-activated receptor-1 and the conversion of proMMP-9 to active MMP-9. In ICH, which was induced in mice by the intracerebral injection of autologous blood, significant areas of brain damage, neuronal death, microglia/macrophage activation, and neutrophil accumulation occurred by 24 h of injury. Importantly, these neuropathological features were reduced in MMP-9 null mice compared with wild-type controls, and the concordant antagonism of thrombin using hirudin also alleviated the injury found in MMP-9 null mice. Our collective results demonstrate that thrombin and MMP-9 collaborate to promote neuronal death in culture and in ICH. To improve the prognosis of ICH, the neurotoxic actions of thrombin and MMP-9 must be inhibited early and simultaneously after injury.

Targeting MMPs in Acute and Chronic Neurological Conditions

“…44 MMP-9 may contribute to the disruption of the blood-brain barrier, 43 aside from its ability to kill neurons. 77 In correspondence with these detrimental outcomes of MMPs early after SCI, the inhibition of MMP activity with GM6001 in the first three days of injury resulted in improvement in functional scores from 3 to 42 days after. 43 Interestingly, the same group reported that if the MMP inhibitor was used for the first seven days, rather than three days, long-term recovery of mice from SCI was attenuated.…”

“…For example, thrombin and MMP-9 combine to mediate the killing of neurons in the mouse brain in vivo, facilitated in part by the activation of MMP-9 by thrombin. 77 The interaction of MMPs with chemokines to alter trafficking of leukocytes into tissues has demonstrated the convergence of functions of two major classes of molecules (FIG. 1).…”

Src kinase inhibition decreases thrombin-induced injury and cell cycle re-entry in striatal neurons

“…Four coronal sections, approximately +2.0 mm to 0 mm from bregma, were cut on a freezing microtome and stored at −20°C in a cryoprotectant composed of 30% glycerol, 20% ethylene-glycol, and 50% PBS. Following three PBS rinses, the brain sections were stained using Hematoxylin & Eosin (Xue et al, 2006). These sections were scanned and analyzed with MCID elite software (Imaging Research, Linton, England).…”

“…ICH activates thrombin, which is a major contributor to ICH induced brain injury (Xi et al, 2003a, Xi et al, 2006. Though thrombin is neuroprotective at low concentrations (Vaughan et al, 1995, Donovan and Cunningham, 1998, Striggow et al, 2000, Friedmann et al, 2001, Xi et al, 2003b, it is detrimental at high concentrations (Donovan et al, 1997, Xi et al, 2003a, Xue et al, 2006, Fujimoto et al, 2007. Direct infusion of 20U of thrombin into rat brain causes a lesion with brain edema formation and neuronal death (Fujimoto et al, 2007).…”

“…Inhibiting protease-activated receptors (PARs) decreases thrombin-induced brain injury (Xue et al, 2006). However, PAR inhibitors may not be good treatment targets for ICH since these might affect the clotting and hemostatic functions of thrombin needed to halt progression of ICH (Thomas and Brugge, 1997).…”