## 4 Preclinical Studies
### In Vivo Studies  
Despite clinical trials, dogs and cats have also been used in research as animal models. The clinical and histopathological similarity of small animals' tissues and neoplasms to humans makes them relevant models for studying treatments in veterinary and medicine. Researchers are dedicated to developing photosensizers that can be extended to clinical use in human medicine [24; 34; 35; 74; 127; 128] and with possible applications in veterinary oncology. This section describes the studies where PDT was applied to health animals used as preclinical models. Porfimer sodium was studied in a canine animal model of glioma obtained by surgical implantation of glioma cells in the brain. The animals received the photosensitizer intravenously and were irradiated with a laser at 630 nm. Despite tumor cell uptake being four times higher than normal brain tissue, severe unwanted effects were associated with higher doses. Still, a lower dose (0.75 mg/kg) of photosensitizer could eliminate the tumor without permanent brain toxicity [129]. The development of a centering balloon for PDT of esophageal cancer has been studied in a canine model. PDT was based on porfimer sodium. The centering balloon led to minimal damage to the normal esophagus in a dose and energy dependent-manner, as increasing photosensitizer concentration and light energy led to increasing damage [130]. Notwithstanding, exposure to 600 J/cm with a 180\({}^{\circ}\) windowed esophageal balloon severely damaged irradiated areas [131]. The size effect of a 360\({}^{\circ}\) windowed balloon was also assessed in a canine esophageal model. PDT-induced lesions were decreased by increasing the balloon size [132]. The normal canine esophagus has also been exploited to compare continuous wave and pulsed laser light. Both pulsed and continuous wave lasers were used in proximal and distal sites. Regardless of the laser type, mucosal ulceration, acute inflammation, serosal oedema, oedema, hemorrhage, and vascular degeneration with inflammation were observed. Based on the gross and histological examination of the lesions, the continuous and pulsed laser-induced injuries could not be distinguished [81]. Sequential porfimer sodium-based PDT sessions were shown to be safe in 13 dogs. Bladder capacity fully recovered in most cases. However, persistent bladder capacity loss  
Figure 5: Preclinical applications of PDT in in vivo and in vitro models. Created with Biorender.com. was observed after the second session. The histopathologic analysis revealed a superficial focal muscle injury in a dog that received three treatments [133]. Fifteen dogs were submitted to an intrathoracic HPD-based PDT to explore thoracic tissue tolerance. Following the photosensitizer administration, the animals underwent a thoracotomy and red-light irradiation (5 to 40 J/cm2). One week after PDT, the extension of the lung's hemorrhagic lesions increased with an increasing light fluence rate. The higher light doses induced superficial hemorrhagic lesions in the heart and diaphragm. Six months later, complete healing was achieved in the lungs, and fibrotic plaques in the heart and diaphragm were observed. The most unaffected tissue was the chest wall [134]. A canine model of transmissible sarcoma was used to investigate the effects of PDT on malignant tumors and normal tissues. Tumors were developed through canine transmissible sarcoma cells subcutaneous implantation. After tumor development (35 mm in diameter), the animals received HPD (5 mg/kg) intravenously. Irradiation took place with an excimer dye laser at 630 nm. The mean diameter of tumor necrosis rapidly increased in parallel with the increase in total irradiation energy, and, as a side effect, cutaneous tissue showed a deep open ulcer [135]. In an animal model for laryngeal cancer, 11 dogs were given 3 mg/kg of HPD three hours before surgery. Then, varying irradiation amounts were delivered through argon laser (10 to 150 J/cm2 of 630 nm), with simultaneous and serial temperature readings.