N-tosyl-l-phenylalanyl-chloromethylketone reduces hypoxic–ischemic brain injury in rat pups
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.
References (30)
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding
Anal. Biochem.
(1976)Mechanisms of neuronal damage in brain hypoxia/ischemia: focus on the role of mitochondrial calcium accumulation
Pharmacol. Ther.
(1998)- et al.
DNA degradation and proteolysis in thymocyte apoptosis
Toxicol. Lett.
(1995) - et al.
Serine and cysteine proteinase inhibitors prevent nitric oxide production by activated macrophages by interfering with signal transcription of the inducible NO synthase gene
Biochem. Biophys. Res. Commun.
(1995) - et al.
Increase in nitric oxide in the hypoxic–ischemic neonatal rat brain and suppression by 7-nitroindazole and aminoguanidine
Euro. J. Pharmacol.
(1998) - et al.
Protease inhibitors differentially regulate tumor necrosis factor-induced apoptosis, nuclear factor-κB activation, cytotoxicity, and differentiation
Blood
(1995) - et al.
The embryonic cell lineage of the nematode Caenorhabditis elegans
Dev. Biol.
(1983) - et al.
Brief post-hypoxic–ischemic hypothermia markedly delays neonatal brain injury
Brain Dev.
(1997) - et al.
Sensorimotor function and neuropathology five and six weeks after hypoxia–ischemia in seven-day-old rats
Pediatr. Res.
(1997) - et al.
Characterization of post-ischemic behavioral deficits in gerbils with and without hypothermic neuroprotection
Brain Res.
(1998)