The development and application of animal models of thrombosis have played a crucial role in the discovery and validation of novel drug targets and the selection of new agents for clinical evaluation, and have informed dosing and safety information for clinical trials. These models also provide valuable information about the mechanisms of action/interaction of new antithrombotic agents. Small and large animal models of thrombosis and their role in the discovery and development of novel agents are described. Methods and major issues regarding the use of animal models of thrombosis, such as positive controls, appropriate pharmacodynamic markers of activity, safety evaluation, species specificity, and pharmacokinetics, are highlighted. Finally, the use of genetic models of thrombosis/hemostasis and how these models have aided in the development of therapies that are presently being evaluated clinically are presented.

Vigorous anticoagulation with heparin sodium and sodium warfarin singly and in combination did not prevent the margination and endothelial sticking reaction of leucocytes in rabbit ear chambers damaged by heat. The general inflammatory reaction observed in this preparation was similarly uninfluenced by the anticoagulants. An unexpected finding after administration of heparin was the enhanced formation of platelet and fibrin-like thrombi within damaged ear chambers. Sodium warfarin did not induce or prevent this heparin effect. Production of these heparin-associated thrombi was minimized in animals subjected to defibrinogenation in vivo whereas leucocytic sticking was not modified. Although defibrinogenation was not absolute, these experiments represent additional proof that the sticking of white blood cells to vascular endothelium is not causally related to the fibrinogen-fibrin system.

Platelets are intimately involved in hemostasis and thrombosis. Under physiological conditions, circulating platelets do not interact with microvascular walls. However, in response to microvascular injury, platelet adhesion and subsequent thrombus formation may be observed in venules and arterioles in vivo. Numerous intravital video microscopy techniques have been described to induce and monitor the formation of microvascular thrombi. The mechanisms of microvascular injury vary widely among different models. Some models induce platelet activation with minimal effects on endothelium, others induce endothelial inflammation or injury, while other models lead to thrombus formation associated with endothelial denudation. The molecular mechanisms mediating platelet-vessel wall adhesive interactions differ among various models. In some instances, differences in responses between venules and arterioles are described that cannot be explained solely by hemodynamic factors. Several models for induction of microvascular thrombosis in vivo are outlined in this review, with a focus on the mechanisms of injury and thrombus formation, as well as on differences in responses between venules and arterioles. Recognizing these characteristics should help investigators select an appropriate model for studying microvascular thrombosis in vivo.

Venous stasis, injury or alteration of the intima and alterations in the coagulability of the blood are the three most common etiologic factors in thrombophlebitis. Usually at least two of these factors must be present before the clinical manifestations of the disease develop.A plan of treatment based on correcting these three factors has been used in over 250 cases of thrombophlebitis, and it is believed that a significant decrease was brought about in the acute and long term disability and in the occurrence of complications. The program consists of absolute bed rest in a hospital for about a week, elevation of the foot of the bed, administration of anticoagulants and adenosine-5-monophosphate for at least six weeks, progressive ambulation after the fourth day of treatment, with avoidance of prolonged standing and sitting, and adequate elastic support. Treatment must be continued until the patient has returned to full, normal activity and all signs of phlebitis have disappeared.

OBJECTIVE AND DESIGN: Oxidized low-density lipoproteins (oxLDL) and protein fractions obtained by size exclusion chromatography of oxLDL were tested for vascular permeability effects on topical application to the hamster cheek pouch. MATERIALS: The hamster cheek pouch was prepared for intravital microscopy observations of macromolecular leakage at post capillary venules (=leaks) with FITC-dextran as tracer. Treatment: OxLDL (0.1 mg/ml), PAF (platelet activation factor, 50-100 nM) and protein fractions of oxLDL (10 microg/ml) were applied topically to hamster cheek pouches. RESULTS: Application of oxLDL and PAF resulted in reversible increases in the number of leaks. The PAF-antagonist WEB 2170, L-NAME and a beta(2)-adrenoceptor agonist inhibited (P<0.01) almost completely the macromolecular leakage induced with oxLDL or PAF. Protein fractions were found to be more effective than unfractionated oxLDL in inducing plasma leakage as calculated on mg/ml-basis. CONCLUSION: Hamster oxLDL is a potent inducer of macromolecular leakage increase in the hamster cheek pouch microcirculation. The principal effect is mediated by PAF-like structures produced by the oxidation of the LDL-particle but oxLDL also contains low molecular weight proteins that could contribute to the overall vascular permeability increasing effect of ox LDL.

We have studied the role of the immune response in the morphology of the leishmaniotic granuloma induced in the cheek pouch of hamsters, an immunologically privileged site, after inoculation of 3 x 10(5) Leishmania mexicana. Animals were histologically and immunologically evaluated until 120 days after inoculation. Independent of the time of sacrifice, the animals were always non-reactors to the footpad test (FPT). At histology, the introduction of L. mexicana in the cheek pouch leads to an abscess that evolves to a granulomatous reaction rich in amastigote forms, and later it leads to resolution, even in the absence of immune response detectable by FPT. Our results demonstrate that the development of immune response is not preponderant for the control of infection induced by L. mexicana inoculated subcutaneously in the cheek pouch of the hamster. It also suggests that the macrophages present in the leishmaniotic granuloma are capable of eliminating this parasite, even in the absence of immune response evaluated by FPT.

This study presents the results of T. mentagrophytes inoculation in the cheek pouch of the hamster, an immunologically privileged site. Forty two animals were used: 21 inoculated with 10(6) fungi in the cheek pouch (group 1) and 21 inoculated initially with 10(6) fungi in the foot pad and 15 days later in the cheek pouch, with the same amount of fungi (group 2). Animals were sacrificed at 20 hours, 3, 7, 14, 30, 60, and 120 days; samples from inoculated cheek pouch, and foot pads submitted to the foot pad test (FPT), were collected. Independent of group and time of evolution of infection, animals did not develop delayed hypersensitivity evaluated through the FPT. The pre-inoculation of fungi in the foot pad did not change the morphology of lesions induced in the cheek pouch. Therefore, in animals of group 1 and 2, the introduction of the fungus in the cheek pouch resulted in focal lesion composed of a sterile acute inflammatory infiltrate, with abscess formation that evolved to a macrophagic reaction, and later to resolution even in the absence of immune response detectable by FPT. Our results indicate that in spite of the important role of the immune response in the spontaneous regression of dermatophytosis, other factors are also an integral part in the defense against this fungal infection.

OBJECTIVE To evaluate a modified method of carcinogenesis induction using the 9,10-dimethyl-1,2-benzanthracene (DMBA) sustained-release suture technique followed by arecaidine promotion in the hamster cheek pouch model. STUDY DESIGN Prospective, controlled animal study. METHODS Number 3-0 cotton sutures were impregnated with DMBA and coated with silicone elastomer. These sutures were placed in the cheek pouch of Syrian hamsters in the submucosal space to a length of approximately 1.5 cm. The suture placement was confirmed every 2 weeks and replaced if lost. After 12 weeks, the DMBA-coated sutures were removed. The cheek pouches were everted and painted with a solution of arecaidine three times weekly for up to an additional 4 weeks or until the tumor reached a size of 100 mm2. RESULTS We placed sutures in 165 Syrian hamster cheek pouches. Of these, 133 hamsters (80.6%) produced squamous cell carcinomas that reached a size of 100 mm2 and then were randomly selected for treatment in a new drug trial. Twenty-six hamsters (15.8%) were found dead and 6 (3.6%) were killed because of severe inflammation. CONCLUSIONS The DMBA hamster cheek pouch model is a reliable and efficient animal model for inducing squamous cell carcinoma and can be used to study upper aerodigestive tract tumors.

The pharmacokinetics (PK) of the photosensitizer tetra(m-hydroxyphenyl)chlorin (mTHPC) was measured by optical fiber-based light-induced fluorescence spectroscopy (LIFS) in the normal and tumoral cheek pouch mucosa of 29 Golden Syrian hamsters with chemically induced squamous cell carcinoma. Similar measurements were carried out on the normal oral cavity mucosa of five patients up to 30 days after injection. The drug doses were between 0.15 and 0.3 mg per kg of body weight (mg/kg), and the mTHPC fluorescence in the tissue was excited at 420 nm. The PK in both human and hamster exhibited similar behavior although the PK in the hamster mucosa was slightly delayed in comparison with that of its human counterpart. The mTHPC fluorescence signal of the hamster mucosa was smaller than that of the human mucosa by a factor of about 3 for the same injected drug dose. A linear correlation was found between the fluorescence signal and the mTHPC dose in the range from 0.075 to 0.5 mg/kg at times between 8 and 96 h after injection. No significant selectivity in mTHPC fluorescence between the tumoral and normal mucosa of the hamsters was found at any of the applied conditions. The sensitivity of the normal and tumoral hamster cheek pouch mucosa to mTHPC photodynamic therapy as a function of the light dose was determined by light irradiation at 650 nm and 150 mW/cm2, 4 days after the injection of a drug dose of 0.15 mg/kg. These results were compared with irradiations of the normal oral and normal and tumoral bronchial mucosa of 37 patients under the same conditions. The reaction to PDT of both types of human mucosae was considerably stronger than that of the hamster cheek pouch mucosa. The sensitivity to PDT became comparable between hamster and human mucosa when the drug dose for the hamster was increased to 0.5 mg/kg. A significant therapeutic selectivity between the normal and neoplastic hamster cheek pouch was observed. Less selectivity was found following irradiations of normal mucosa and early carcinomas in the human bronchi. The pharmacodynamic behavior of mTHPC was determined by test irradiations of the normal mucosa of hamsters and patients between 6 h and 8 days after injection of 0.5 and 0.15 mg/kg in the hamsters and the patients, respectively. The normal hamster cheek pouch showed a maximum response to irradiation 6 h after injection and then decreased continuously to no observable reaction at 8 days after injection. The reaction of the normal human oral mucosa, however, showed an increasing sensitivity to the applied light between 6 h and 4 days after mTHPC injection and then decreased again at 8 days. The hamster model with the chemically induced early squamous cell cancer in the cheek pouch thus showed some similarity to the early squamous cell cancer of the human oral mucosa considering the PK. However, a quantitative difference in fluorescence signal for identical mTHPC doses as well as a significant difference in pharmacodynamic behavior were also observed. The suitability of this animal model for the optimization of PDT parameters in the clinic is therefore limited. Hence great care must be taken in screening new dyes for PDT of early squamous cell cancer of the upper aerodigestive tract based upon observables in the hamster cheek pouch model.

The subcutaneous tissue of the hamster cheek pouch, a site of immunologic privilege, has been used to investigate the potential infectivity of different types of parasites. It has been demonstrated that the implantation of fragments of lesions induced by the fungus Lacazia loboi, the etiologic agent of Jorge Lobo's disease, into the subcutaneous tissue of the hamster cheek pouch resulted in parasite multiplication and dissemination to satellite lymph nodes16. Here we describe the evolution of lesions induced by the inoculation of the isolated fungus into this immunologically privileged site. The morphology of the inflammatory response and fungal viability and proliferation were evaluated. Inoculation of the fungus into the cheek pouch induced histiocytic granulomas with rare lymphocytes. Although fungal cells were detected for a period of up to 180 days in these lesions, the fungi lost viability after the first day of inoculation. In contrast, when the parasite was inoculated into the footpad, non-organized histiocytic lesions were observed. Langhan's giant cells, lymphocytes and fungal particles were observed in these lesions. Fungal viability was observed up to 60 days after inoculation and non-viable parasites were present in the persistent lesions up to 180 days post-inoculation. These data indicate that the subcutaneous tissue of the hamster cheek pouch is not a suitable site for the proliferation of Lacazia loboi when the fungus isolated from human tissues is tested.

The feasibility of using the hamster cheek pouch/dimethyl-benzanthracene (DMBA) system as an experimental model of lymphatic metastasis was investigated. Forty male Syrian golden hamsters treated with DMBA were divided into two equal groups--one with surgical excision of their tumors and a control group without tumor excision. In the excision group, the animals received three applications/week to the left cheek pouch of 0.3% DMBA in acetone for 14 weeks. Following a three-week observation period, the tumors in the pouch were excised at their base, and the animals were killed after four weeks of further observation. In the control group, the animals were treated for 14 weeks in a manner similar to that used for the excision group, left for seven weeks without treatment, and then killed. Cheek pouches with tumors and cervical lymph nodes were processed for histological examination. All of the animals, both with and without metastasis, had borne squamous cell carcinomas (SCC) in their treated cheek pouches. Histologically, seven out of 16 animals in the excision group showed metastatic deposits of SCC confined to the left cervical lymph nodes, while in the control group, metastasis was not found in any of the 19 animals with SCC in their cheek pouches. The results demonstrate that surgical excision of the hamster cheek pouch carcinoma is efficient in producing unequivocal lymph node metastasis.