A novel excisional wound pain model for evaluation of analgesics in rats.

BACKGROUND: Management of pain from open wounds is a growing unmet healthcare need. However, the models available to study pain from wounds or to develop analgesics for the patients suffering from them have primarily relied on incisional models. Here, we present the first characterized and validated model of open wound pain. METHODS: Unilateral full-skin excisional punch biopsy wounds on rat hind paws were evaluated for evoked pain using withdrawal responses to mechanical and thermal stimulation, and spontaneous pain was measured using hind paw weight distribution and guarding behavior. Evaluations were done before wounding (baseline) and 2-96 hours post-wounding. The model was validated by testing the effects of buprenorphine and carprofen. RESULTS: Pain responses to all tests increased within 2 hours post-wounding and were sustained for at least 4 days. Buprenorphine caused a reversal of all four pain responses at 1 and 4 hours post-treatment compared to 0.9% saline (P < 0.001). Carprofen decreased the pain response to thermal stimulation at 1 (P ≤ 0.049) and 4 hours (P < 0.011) post-treatment compared to 0.9% saline, but not to mechanical stimulation. CONCLUSIONS: This is the first well-characterized and validated model of pain from open wounds and will allow study of the pathophysiology of pain in open wounds and the development of wound-specific analgesics. Key Words.

The critical role of the intrinsic VSMC proliferation and death programs in injury-induced neointimal hyperplasia.

Postangioplasty and in-stent restenosis remain ominous problems in percutaneous coronary intervention where good animal models of restenosis proneness and resistance are needed. We accidentally discovered that the carotid arteries (CAs) of the Harlan and Sasco substrains of Sprague-Dawley rats display drastically different restenosis phenotypes following balloon-induced endothelial denudation. When subjected to balloon injury, Sasco CAs exhibited significantly larger neointimal mass than did Harlan CAs at both days 14 and 32, as evidenced by a higher intima-to-media ratio and a greater number of intimal cells in Sasco CAs. This was due to a greater cell proliferation and to a less vigorous apoptosis of Sasco neointima, as assessed by 5-bromo-2'-deoxyuridine and terminal deoxynucleotidyl transferase-deoxyuridine nick-end labeling staining, respectively. At a cellular level, whereas vascular smooth muscle cells (VSMCs) isolated from Sasco and Harlan CAs were identical in morphology and in propensity to migrate, Sasco VSMCs proliferated more robustly and died far less, suggesting that under the exact same microenvironment, Sasco and Harlan VSMCs respond to growth and noxious stimuli in a drastically different fashion and that Sasco's significantly more robust neointimal proliferation after vascular injury in vivo can be accounted for by these intrinsic differences in VSMCs of these substrains in vitro. Sasco and Harlan Sprague-Dawley rats as well as VSMCs from these rats will prove to be powerful tools to study genes involved in the pathogenesis of restenosis.

Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2.

Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.