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Jul 26, 2011

Studies Show Natural Protein May Provide Benefits Against Stroke up to 12 Hours After Onset

  • Scientists suggest that treating stoke patients with an endogenous immunomodulatory neuroprotectant protein, αB-crystallin (Cryab), could help limit brain damage even if first administered 12 hours after the onset of stroke. Studies by a Stanford University School of Medicine team in a mouse model of stroke found that administering Cryab to animals 12 hours after stroke induction led to significantly reduced stroke volume and lower levels of inflammatory cytokines associated with stroke pathology than untreated mice.

    Conversely, animals in which the Cryab gene had been knocked out demonstrated increased lesion size and diminished neurologic function after stroke than wild-type mice. The research team, led by Gary K. Steinberg, M.D., director of Stanford’s Institute for Neuro-Innovation and Translational Neurosciences, reports its findings in PNAS in a paper titled “Systemic augmentation of αB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation.”

    Tissue plasminogen activator (tPA) is currently the only FDA-approved treatment for stroke, and must be given within 4.5 hours of stroke onset to be effective, the researchers note. tPA acts through the conversion of plasminogen into active plasmin, to cleave the blood clot, and exemplifies strategies that aim to alter the obstructive blood clot rather than actually protect the damaged brain.

    Cryab, meanwhile, is a small heat shock protein (designated sHSP B5) that is constitutively expressed in the lens of the eye and muscle, and is induced in many types of brain injury. Previous studies have shown the protein has both anti-apoptotic and immunomodulatory properties; it is also the most abundant induced transcript in multiple sclerosis lesions and is highly expressed in areas of inflammation, the authors add.

    Previous studies by a group led by co-author Lawrence Steinman, M.D., George A. Zimmermann professor of neurology and neurological sciences and pediatrics at Stanford, had found that Cryab reduces the severity of brain damage caused by multiple sclerosis, and that the protein can also limit the damage caused by reduced blood supply to heart tissue and to the retina. These findings, coupled with other research suggesting that Cryab has neuroprotective properties, led Drs. Steinberg, Steinman, and colleagues to evaluate the effects of Cryab deficiency on cerebral ischemia.

    They found that Cryab-knockout animals demonstrated significantly larger lesions two days after an induced cerebral artery occlusion than wild-type mice. This increased damage in the Cryab-deficient animals persisted at seven days, indicating that Cryab deficiency affects both the early and delayed phases of ischemic damage. The Cryab-knockout mice also scored significantly worse in terms of functional assessment.

    Interestingly, granulocyte and macrophage populations, and the subpopulation of monocytes, were significantly higher in the Cryab-deficient mice at the two-day post-stroke stage, but not at seven days. The numbers of lymphoid cells, however, were significantly increased in the knockout animals at seven days. More specifically, there were increased numbers of granulocytes and activated macrophages in the Cryab-deficient mice than in the wild-type controls at two days. There were also marked increases in the numbers of T cells in the brains of Cryab knockouts than wild-type mice at seven days and, in particular, increased levels of IL-17-producing γδ-TCR+ (γδ-T) cells. The overall data suggested that “a deficiency of Cryab might lead to a more vigorous inflammatory response to stroke,” the authors note.

    Because some research has previously suggested that Cryab expression is upregulated in neurons and astrocytes after cerebral ischemia, the researchers moved on to analyze levels of Cryab in the plasma of wild-type mice before stroke, and at various time points after the induction of stroke. This showed that Cryab levels were significantly increased at the 12 hour time point, and decreased gradually over the next seven days.

    When they analyzed the plasma concentrations of Cryab in human patients with ischemic stroke, they found increased levels of the protein in younger patients within four hours of stroke symptom onset, but surprisingly not in older patients. In younger patients only, lesion volume also correlated with plasma Cryab levels, “perhaps indicating that the body’s endogenous response to stroke is age dependent,” the team suggests.

    To test the notion that increased plasma Cryab after stroke is beneficial, Cryab-knockout mice were given intraperitoneal injections of recombinant Cryab protein, starting one hour before stroke onset and continuing at 12 hours, 24 hours, and then daily for seven days in total. Lesion sizes in the Cryab-treated knockout mice were significantly smaller than those of the knockout animals that weren’t given exogenous Cryab. Evaluation of splenocytes from PBS-treated wild-type mice, Cryab-deficient animals, and Cryab-treated Cryab-knockout animals showed that those of PBS-treated knockouts produced more proinflammatory IL-2, IL-17, and TNF than both PBS-treated wild-type mice and Cryab-treated Cryab-deficient mice. The animals receiving exogenous Cryab also produced more anti-inflammatory IL-10. “These data indicate that restoration of plasma Cryab by systemic treatment modulates the peripheral inflammatory response and is sufficient to decrease the lesion sizes in Cryab−/− mice to the levels of wild-type mice after stroke,” the researchers note.

    They progressed to evaluate whether Cryab therapy could benefit wild-type mice after stroke onset. When Cryab was administered one hour before and 12 hours after stroke onset, the lesion size at two days was not different between PBS- and Cryab-treated wild-type mice groups. In contrast, when it was administered 1 hour before, 12 hours and 24 hours after, and then daily for seven days, the lesion sizes were significantly reduced in the Cryab-treated group compared with the PBS-treated group. “Moreover, starting the initial treatment even 12 hours after the stroke onset—making the treatment highly relevant if translated into the clinic—conferred neuroprotection in the Cryab-treated group,” the team claims. Analysis of splenocytes cytokines seven days after stroke in wild-type mice again showed that the Cryab-treated animals produced less proinflammatory IL-2, IL-17, IFN-γ, IL-12p40, and IL-6, and more anti-inflammatory IL-10, than the PBS-treated animals.

    “Our findings describe a therapeutic role for Cryab in stroke, and emphasize how it functions as an endogenous neuroprotectant by modulating the immune system,” the authors conclude. “Its presence as an endogenous protectant can be exploited by administering it in larger quantities as a therapeutic agent. Its benefit seen with starting the treatment 12 hours after stroke would represent a significant improvement over tPA if translated to the clinic.”


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