tPA Variant tPA-S481A prevents impairment of cerebral autoregulation during hypotension and histopathology

after TBI

William M. Armstead, John Riley, Serge Yarovoi, Douglas B. Cines, Douglas H. Smith,

and Abd Al-Roof Higazi Departments of Anesthesiology and Critical

Care, Pharmacology, Pathology, and Neurosurgery

University of Pennsylvania

Impaired Cerebral Autoregulation

MAP or CPP

CB

F

(m l/1

00

g/

mi

n)

Intact Cerebral Autoregulation

MAP or CPP

CB

F

(ml/1

00g

/m

in)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15

GCS at the time of autoregulation testing

Lowest ARI

Relationship between GCS and cerebral autoregulation (lARI)

The ARI is the percent change in estimated cerebrovascular resistance (eCVR) per percent change in CPP: ARI = % eCVR / % CPP The eCVR is the ratio of CPP to Vmca. Thus, an ARI of 0 represents absent autoregulation (pressure dependent Vmca), while an ARI of 1.0 represents perfect autoregulation. Autoregulatory capacity was considered intact if the ARI was ³ 0. 4.

Freeman, Udomphorn, Armstead, Fisk, Vavilala Anesthesiology 108: 588-595, 2008.

Impairment of autoregulation correlates with GCS

Cerebral autoregulation (ARI) is more impaired after moderate-severe TBI in children < 4 years.

0.001233ARI > 0 (intact autoregulation)

58ARI = 0 (absent autoregulation)

0.65 ± 0.310.33 ± 0.32Mean ARI

pAge 4 ( 12 ± 3) years ≥(n=27)

Age < 4 (2 ± 1) years (n=11)Table 2:

Glasgow outcome score (6 month GOS) is worse after moderate-severe TBI in children < 4 yrs.

0.005173Age > 4 years

67Age < 4 years

p6 month GOS > 4 (n=23)

6 month GOS < 4 (n=10)

Table 4:

Freeman, Udomphorn, Armstead, Fisk, Vavilala Anesthesiology 108: 588-595, 20008

Plasminogen activators, NMDA, and outcome in TBI

• Glutamate release after TBI contributes to impaired cerebral hemodynamics and histopathology.

• Tissue plasminogen activator (tPA) enhances excitotoxic neuronal cell death through interactions with NMDA receptors.

• tPA upregulation post TBI contributes to impaired cerebral hemodynamics, histopathology, and aggravation of NMDA-mediated impairment of cerebral hemodynamics, including disturbed autoregulation during hypotension.

tPA contributes to impaired NMDA cerebrovasodilation through activation of JNK and ERK MAPK. Neurologic Res, in press.

Impaired NMDA receptor mediated cerebrovasodilationcontributes to disturbed autoregulation during hypotensionafter TBI. Develop Brain Res 139: 19-28, 2002.

Purpose

tPA-S481A is a catalytically inactive tPA variant with single mutation in the active site that maintains its docking site and capacity to bind to the NMDA receptor, but cannot cleave/activate the receptor. This study investigated the ability of tPA-S481A to prevent dysregulation and histopathology after TBI.

Hypothesis

Generation of a mutant tPA that competes with wildtype tPA for binding to NMDA receptors andprotects it from cleavage/activation by wild typetPA will improve outcome after TBI.

In the context of the neurovascular unit, impairedcerebral hemodyanamics contributes to outcome.

General Methodology• Combined physiologic, biochemical, pharmacologic approach towards study

of cerebral hemodynamics in piglet model.• CBF determined by microspheres, TCD, DCS• Immunohistochemistry. histopathology

Advantages of the piglet model 1-5 day old pig 1-2 yr old child≈ gyrencephalic brain more white than grey matter selective vulnerability of white matter

FPI produces pial artery vasoconstriction, which was Blocked by tPA-S481A (1 mg/kg iv) 30 min post injury

Pial artery dilation during hypotension blunted after FPI,but tPA-S481A prevented such impairment.

FPI was associated with marked neuronal cell loss in CA1and CA3 hippocampus, which was prevented by tPA-S481A

Conclusions

• These data indicate that tPA-S481A prevents impairment of cerebral autoregulation during hypotension and histopathology after FPI via its ability to bind but not activate the NMDA receptor.

• Use of this tPA variant is a novel approach towards limiting toxicity of NMDA receptor activation associated with the robust increase in tPA and glutamate within the brain after TBI.

Funding: RO1 NS53410, RO1 HD57355