D.Phil. Faculty of Health Sciences, University of the Witwatersrand, 2009 / In this thesis I describe the development of an animal model of sustained hyperalgesia induced by exposure to ultraviolet (UV) A light to the rat’s tail, and the role of the Cfibre barrage and peripheral afferent fibre sensitization in this model of hyperalgesia. Exposure of rats’ tails to UVA-light caused hyperalgesia to a noxious thermal challenge, immersion of the rats’ tails into 49°C water, and a noxious mechanical challenge, application of a static force of 3.9N by a bar algometer onto the rats’ tails. The hyperalgesia to the thermal challenge lasted eight days and hyperalgesia to the mechanical challenge continued for up to 16 days. Despite the sustained hyperalgesia, rats exposed to UVA-light showed no overt signs of morbidity as they gained weight normally and were mobile throughout the study. Histological examination of rat tail tissue showed mild, chronic inflammation in rats exposed to UVA-light and in rats that had their tails covered with a protective layer of aluminium foil during UVA-light exposure. This inflammation was therefore not responsible for the behavioural hyperalgesia. To investigate the role of C-fibre barrage in the development of hyperalgesia after UVA-light exposure, I pre-emptively blocked C-fibre activation during UVA-light exposure with the local anaesthetic bupivacaine. Injection of bupivacaine (1ml of 0.5%), into the base of the tail prevented the development of thermal hyperalgesia to tail immersion in 49°C water. However, it did not prevent the development of hyperalgesia to a noxious punctate challenge. Thus the sustained mechanical hyperalgesia did not depend on the activation of the C-fibre barrage, but thermal hyperalgesia did depend on the activation of a C-fibre barrage during the conditioning event of UVA-light exposure. Lastly, in rats anaesthetised with enflurane, I examined the responses of coccygeal primary afferent fibres to noxious thermal and mechanical stimulation after UVA-light exposure of their receptive fields on the tail. I investigated only pure nociceptive afferents and ignored those afferents that responded to challenges in the noxious and non-noxious ranges. The peak firing rates and areas under the curve of post-challenge histograms, a measure of neuronal firing over time, of Ad- and C-fibres were increased when noxious blunt and punctate challenges were applied to the rats’ tails after UVA-light exposure, showing that Ad- and C-fibres that encode for noxious mechanical challenges were sensitized. The peak firing rate of C-fibres that were responsive to noxious thermal challenges were not increased after UVA-light exposure. Therefore, thermal hyperalgesia was probably mediated by sensitization of central nervous system neurones. In summary, I developed a model of sustained mechanical and thermal hyperalgesia caused by UVA-light exposure of the rat tail. The thermal hyperalgesia was initiated by the C-fibre barrage, while mechanical hyperalgesia did not depend on the C-fibre barrage and peripheral afferent sensitization of Ad- and C-fibres could account for the mechanical hyperalgesia.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Reloading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HFI) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2-6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months followed by 3% copper hydroxide carbonate [CuC03»Cu(0H)2] / IT2018

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Fe- loading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HH) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2 - 6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months / IT2018

Assegurar o bem-estar dos animais de laboratório e evitar a dor e o sofrimento desnecessários são as principais considerações na experimentação. Por isso, a investigação em busca de novos protocolos anestésicos que garantam um mínimo ou nulo desconforto dos animais tem-se convertido num tema prioritário. Neste estudo realizou-se a comparação do efeito da combinação de xilazina (X) e ketamina (K) com acepromazina (A) e opióides [metadona (Me), morfina (Mo) e tramadol (T)] com a finalidade de avaliar sua influência nos parâmetros fisiológicos de ratos de laboratório, para ambos os sexos, bem como seu poder analgésico e o efeito da oxigenação sobre os mesmos. A associação XKA para ratos machos e fêmeas e a associação XKMe para fêmeas foram as mais seguras e eficazes para procedimentos anestésicos. Porém, os resultados do teste de formalina com a medição da vocalização ultrassônica (VUS) sugerem que o protocolo de XKA tem um poder analgésico baixo, não sendo indicado para procedimentos que possam gerar dor moderada ou severa. Todas as associações anestésicas tiveram efeitos importantes como diurese, manutenção dos olhos abertos e hiperglicemia, os quais devem ser considerados quando possam influenciar nos resultados experimentais. Também, se conseguiu demonstrar que a oxigenação melhora a saturação de oxigênio (SO2) e os valores da pressão parcial de oxigênio (pO2) confirmando que sua utilização deveria ser sempre parte dos procedimentos experimentais com anestesia injetável para evitar a hipoxemia. Não obstante, observou-se uma acidose respiratória por aumento da pressão parcial do CO2 (pCO2) e diminuição do pH, cuja causa se relacionou à hipoventilação por depressão respiratória e acúmulo de CO2 durante o transcorrer da anestesia. Tal situação demonstra a necessidade de oxigenar os animais desde a indução da anestesia e de administrar medicamentos para reverter a depressão respiratória como a naloxona, bem como utilizar animais que não apresentem nenhum tipo de comprometimento respiratório. Portanto, a inclusão de analgesia e a oxigenação nos protocolos anestésicos injetáveis devem ser utilizadas de forma rotineira garantindo a mínima presença de dor e, com isso, resultados mais confiáveis nos procedimentos experimentais. / Ensuring the wellbeing of laboratory animals and avoiding unnecessary pain and suffering is one of the main considerations in experimentation. Therefore, the investigation of new anesthetic protocols that guarantee a minimum or null discomfort of the animals has become a priority theme. In this study, it was made an evaluation of the effect of the combination of xylazine (X) and ketamine (K) with acepromazina (A) and opioids [methadone (Me), morphine (Mo) e tramadol (T)] with the purpose of comparing their influence on the physiological parameters of laboratory rats, for both sexes, and evaluating their analgesic power and the effect of the oxygenation on them. The XKA protocol for male and females rats and the XKMe protocol for females were the safest and most effective for anesthetic procedures. However, the results of the formalin test with the measurement of the ultrasonic vocalization (VUS) suggest that the XKA protocol had a low analgesic power, and it is not indicated for procedures that can generate moderate or severe pain. All anesthetic protocols had important effects as diuresis, maintenance of open eyes, and hyperglycemia; these effects should be considered when they could influence in the experimental results. It was demonstrated that oxygenation improves oxygen saturation (SO2) and oxygen partial pressure (pO2) confirming that its use should be considered in the experimental procedures with injectable anesthesia to avoid hypoxemia. Nevertheless, a respiratory acidosis was observed due to the increase in partial pressure of CO2 (pCO2) and the decrease in pH, that could be caused for the hypoventilation due to respiratory depression and CO2 accumulation during the course of anesthesia. This leads to consider the need to pre-oxygenate animals from induction, to use drugs to reverse respiratory depression such as naloxone, and to work with animals that are not respiratory compromised. Therefore the inclusion of analgesia and oxygenation in anesthesia protocols should begin to be used routinely ensuring minimal presence of pain and thus more reliable results in the experimental procedures.

Assegurar o bem-estar dos animais de laboratório e evitar a dor e o sofrimento desnecessários são as principais considerações na experimentação. Por isso, a investigação em busca de novos protocolos anestésicos que garantam um mínimo ou nulo desconforto dos animais tem-se convertido num tema prioritário. Neste estudo realizou-se a comparação do efeito da combinação de xilazina (X) e ketamina (K) com acepromazina (A) e opióides [metadona (Me), morfina (Mo) e tramadol (T)] com a finalidade de avaliar sua influência nos parâmetros fisiológicos de ratos de laboratório, para ambos os sexos, bem como seu poder analgésico e o efeito da oxigenação sobre os mesmos. A associação XKA para ratos machos e fêmeas e a associação XKMe para fêmeas foram as mais seguras e eficazes para procedimentos anestésicos. Porém, os resultados do teste de formalina com a medição da vocalização ultrassônica (VUS) sugerem que o protocolo de XKA tem um poder analgésico baixo, não sendo indicado para procedimentos que possam gerar dor moderada ou severa. Todas as associações anestésicas tiveram efeitos importantes como diurese, manutenção dos olhos abertos e hiperglicemia, os quais devem ser considerados quando possam influenciar nos resultados experimentais. Também, se conseguiu demonstrar que a oxigenação melhora a saturação de oxigênio (SO2) e os valores da pressão parcial de oxigênio (pO2) confirmando que sua utilização deveria ser sempre parte dos procedimentos experimentais com anestesia injetável para evitar a hipoxemia. Não obstante, observou-se uma acidose respiratória por aumento da pressão parcial do CO2 (pCO2) e diminuição do pH, cuja causa se relacionou à hipoventilação por depressão respiratória e acúmulo de CO2 durante o transcorrer da anestesia. Tal situação demonstra a necessidade de oxigenar os animais desde a indução da anestesia e de administrar medicamentos para reverter a depressão respiratória como a naloxona, bem como utilizar animais que não apresentem nenhum tipo de comprometimento respiratório. Portanto, a inclusão de analgesia e a oxigenação nos protocolos anestésicos injetáveis devem ser utilizadas de forma rotineira garantindo a mínima presença de dor e, com isso, resultados mais confiáveis nos procedimentos experimentais. / Ensuring the wellbeing of laboratory animals and avoiding unnecessary pain and suffering is one of the main considerations in experimentation. Therefore, the investigation of new anesthetic protocols that guarantee a minimum or null discomfort of the animals has become a priority theme. In this study, it was made an evaluation of the effect of the combination of xylazine (X) and ketamine (K) with acepromazina (A) and opioids [methadone (Me), morphine (Mo) e tramadol (T)] with the purpose of comparing their influence on the physiological parameters of laboratory rats, for both sexes, and evaluating their analgesic power and the effect of the oxygenation on them. The XKA protocol for male and females rats and the XKMe protocol for females were the safest and most effective for anesthetic procedures. However, the results of the formalin test with the measurement of the ultrasonic vocalization (VUS) suggest that the XKA protocol had a low analgesic power, and it is not indicated for procedures that can generate moderate or severe pain. All anesthetic protocols had important effects as diuresis, maintenance of open eyes, and hyperglycemia; these effects should be considered when they could influence in the experimental results. It was demonstrated that oxygenation improves oxygen saturation (SO2) and oxygen partial pressure (pO2) confirming that its use should be considered in the experimental procedures with injectable anesthesia to avoid hypoxemia. Nevertheless, a respiratory acidosis was observed due to the increase in partial pressure of CO2 (pCO2) and the decrease in pH, that could be caused for the hypoventilation due to respiratory depression and CO2 accumulation during the course of anesthesia. This leads to consider the need to pre-oxygenate animals from induction, to use drugs to reverse respiratory depression such as naloxone, and to work with animals that are not respiratory compromised. Therefore the inclusion of analgesia and oxygenation in anesthesia protocols should begin to be used routinely ensuring minimal presence of pain and thus more reliable results in the experimental procedures.

Male mammals typically outperform females in tests of spatial ability. However, in laboratory rats (Rattus norvegicus), from which the majority of data in support of this difference come, sex differences are not consistently found. Since stress affects cognition in males and females differently, I investigated possible sources of stress (e.g. housing conditions, spatial tasks) and the impact they have on cognitive performance in male and female rats. Firstly, I investigated whether isolation housing, purported to be chronically stressful, affected the presence of sex differences in a dark-eyed and an albino strain of rat. Irrespective of sex and strain, I found that young or old rats isolated for long or short periods were not behaviourally or cognitively impaired relative to pairhoused conspecifics. I found, however, that behaviour caused by the acute stress of the task impeded performance. Furthermore, sex differences in performance were found only when the females were more stressed than the males during testing. Additionally, the degree to which the rats found the task stressful depended upon the age at which they travelled from the breeding establishment. In the dark-eyed strain, males were always less stressed than the females, but also out performed the females only if they travelled while young (4-5 weeks old). Both sexes seemed to be less stressed by the task if the rats travelled as adults. Conversely, in the albino strain, males outperformed females only if the rats travelled as adults, because in the young travellers both sexes were equally and highly stressed during testing. Therefore, the acute stress response, which seems to underlie sex differences in cognitive performance, was influenced by the age at which the rats travelled in a sex and strain dependent manner. Next, I considered the impact of the physical attributes of the home cage on a rat’s welfare and performance in a cognitive task. I found that, male and female rats housed with a barrier that reduced visual contact from their cage showed higher levels of behavioural stress in their home cage than did rats housed without a barrier between the cages. Rats housed with the barrier were also more stressed during spatial testing and had poorer cognitive performance relative to rats housed without the barrier. Pair housing did not ameliorate the effect of the barrier. Based on these data, although a rather unorthodox suggestion, I propose that single housing with a view may be preferable to pair housing without a view. One implication of this finding is that the number of animals used in an experiment could be significantly reduced if the home cages allow sufficient visual interactions. Lastly, I investigated the impact of environmental enrichment on spatial cognition and behavioural stress responses. I found, contrary to current opinion, that enriched rats outperformed non-enriched animals not because they had superior cognitive ability but because their behavioural stress response was reduced significantly during testing. Furthermore, withdrawing enrichment from rats for at least one week did not increase stress responses during testing or impair cognitive performance. Therefore, exposure to enrichment, even if later withdrawn, improves welfare by reducing stress during cognitive testing. In conclusion, a differential behavioural stress response during cognitive testing may explain why males outperform females and why enriched animals do better than non-enriched animals in tests of spatial cognition. Furthermore, variation in this behavioural stress response may in part explain why sex differences in performance are not consistently found in laboratory rats.

This chapter begins by describing the appearance of motor abilities, social and play behavior, sexual maturity, and sensory function in laboratory rats. It then discusses cognitive function, the development of simple stimulus associations made in conditioned taste aversion, and complex associations necessitated in spatial memory. The development of brain structures and their possible role in these behaviors are also discussed.