N-tosyl-l-phenylalanyl-chloromethylketone reduces hypoxic–ischemic brain injury in rat pups

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Abstract

N-tosyl-l-phenylalanyl-chloromethylketone (TPCK) in vitro blocks apoptotic pathways leading to cell death. We wished to see if TPCK would reduce brain injury in vivo. Seven-day-old rat pups had the right carotid artery ligated and then received either vehicle or TPCK (5 to100 mg/kg i.p.). They were then given 8% oxygen for 2.25 h. Twenty-two days later, the cerebral hemispheres were weighed to determine the reduction in size in the right hemisphere. TPCK decreased the reduction in right hemisphere weight from 15±3% (vehicle, n=20), to 4±2% (10 mg/kg, n=19, P<0.01). TPCK reduced the number of cells staining for DNA breaks 3 days after injury from 1729±275 mm−2 (vehicle, n=8) to 550±236 mm−2 (10 mg/kg TPCK, n=9, P<0.01), decreased the amount of DNA fragmentation 3 days after injury by gel electrophoreses (20 mg/kg, n=16, P<0.01) and eliminated the increase in nitric oxide metabolites 6 h after injury (vehicle 1.5±0.4, n=10; and 20 mg/kg TPCK 0.0±0.1 nM/mg protein, n=10, P<0.001). TPCK pretreatment in the newborn rat model of hypoxic–ischemic brain injury reduces DNA fragmentation, nitric oxide production and brain injury.

Introduction

Apoptosis describes the programmed death of cells that are no longer needed by the embryo during development (Sulston et al., 1983). Necrotic cell death is the rapid death of cells after injury. However, a subset of injured cells in some protocols follows a pattern of delayed cell death that morphologically and biochemically resemble apoptosis (Ramachandra and Studzinski, 1995). Cytoplasmic proteases play an important role in the pathways leading to apoptotic cell death Fearnhead et al., 1995, Higuchi et al., 1995. Intranuclear DNA fragmentation into low molecular weight segments (DNA laddering) is important evidence for an apoptotic pattern of cell death (Ramachandra and Studzinski, 1995). Terminal deoxynucleotidyl transferase mediated dUTP biotin nick end-labeling (TUNEL staining), although less specific, provides in situ evidence for DNA fragmentation (Ramachandra and Studzinski, 1995).

N-tosyl-l-phenyalanyl-chloromethylketone (TPCK) is a serine protease inhibitor, originally developed as an irreversible chymotrypsin inhibitor (Schoellmann and Shaw, 1993). TPCK is lipid soluble and enters cells easily. It inhibits apoptotic DNA laddering Takauji et al., 1996, Dong et al., 1997, Mansat et al., 1997 and cell death in a number of different cell culture systems. As a cell permeate serine protease inhibitor, TPCK has proved useful in determining the chemical pathways involved in DNA laddering Takauji et al., 1996, Dong et al., 1997, Mansat et al., 1997. TPCK also reduces inducible nitric oxide synthetase production in macrophages (Griscavage et al., 1995) by inhibiting nuclear factor κB (NF-κB) activation (Ruetten and Thiemermann, 1997). TPCK has recently been shown to reduce hippocampal injury in an adult gerbil model of severe forebrain ischemia (Hara et al., 1998). Hypoxic–ischemic brain injury is an important cause of death and disability in human newborns. The developmental stage of the brain of the 7-day-old rat pups resembles that of a human newborn (Palmer et al., 1990). Newborns, who are just leaving the normal physiologic period of neuronal apoptosis, might well have more active apoptotic pathways after cell injury than adults. We hypothesized that treatment with TPCK would reduce hypoxic–ischemic brain injury in newborns.

Section snippets

Animal protocol

This protocol was approved by our institutional committee on animal use. Rats were cared for in accordance with National Institutes of Health guidelines. Using the well-characterized Rice et al. (1981) immature rat hypoxic–ischemic brain Injury model, 7-day-old Sprague–Dawley rat pups were anesthetized with isoflurane and had the common right carotid artery isolated from the nerve and vein, ligated and divided. The pups were returned to their dam for at least 3 h recovery after surgery. Pups

Results

Gross neuropathologic damage was scored 22 days after injury. Nine percent (2/22) of the vehicle-treated pups died prior to 22 days. Five percent (1/21) of the pups given 5 mg/kg of TPCK, 5% (1/20) of the pups given 10 mg/kg, and 13% (3/23) of the pups given 20 mg/kg died prior to 22 days (P=ns, vs. vehicle). Sixty-seven percent (6/9) of the pups given 50 mg/kg of TPCK (P<0.01 vs. vehicle) and 100% of the seven pups given 100 mg/kg died prior to 22 days (P<0.01 vs. vehicle). Most of the deaths

Discussion

TPCK given prior to injury reduces the degree of injury in the focal ischemia model of the neonatal rat pup. Results are similar to that reported in the adult gerbil global ischemia model (Hara et al., 1998). Since delayed neuronal injury sometimes requires a prolonged period to develop (Trescher et al., 1997), we extended the time from injury to brain assessment from the 96 h used in Hara's study in gerbils to 22 days without a reduction in efficacy. Since late hypothermia can effect study

Acknowledgements

This study was funded with an unrestricted grant from Burroughs Wellcome.

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