Apoptosis can be defined as a carefully regulated process, characterized by specific morphologic and biochemical features. It is initiated by both physiologic and pathologic stimuli, and its full ...expression requires a signaling cascade in which caspase activation plays a central role. Knockout mice lacking key genes encoding proteins constituting the core apoptotic cascade have helped us to establish the functional hierarchy of the mechanisms controlling apoptosis in animal development and, to a lesser extent, in disease. Induced mutant mice have also revealed the intimate crosstalk between apoptotic and other homeostatic pathways and have defined distinct temporal and tissue-specific roles of individual apoptotic effectors. Eliminating genes controlling caspase-dependent apoptosis can convert an apoptotic phenotype to a necrotic one, both in vitro and in vivo. This suggests that necrosis and apoptosis represent morphologic expressions of a shared biochemical network through both caspase-dependent mechanisms as well as non-caspase-dependent effectors such as cathepsin B and apoptosis-inducing factor. The cell death program, whether by apoptosis or necrosis, is mediated through an integrated cascade, which can be accessed at multiple sites, and propagated through numerous branch points. An understanding of the physiologic conditions that influence these decisions is required to adequately prevent, or induce, cell death.
Age-related macular degeneration (AMD) is a degenerative condition that begins in Bruch’s membrane and progresses to involve the retinal pigment epithelium and ultimately the overlying ...photoreceptors. The only required etiologic factor is age, and AMD is regarded as the leading cause of blindness in individuals older than 65 years. AMD results from variable contributions of age, environment, and genetic predisposition. Many loci are linked to AMD; in the majority of cases, the disease is associated with polymorphisms within these genes, rather than mutations that ablate gene function. The etiologic complexity of AMD is reflected by the paucity of animal models that entirely replicate the human disease. This review compares the salient anatomy of the primate and rodent retina, particularly in the light of AMD pathology. It next discusses prevailing hypotheses explaining how AMD may develop. These include the role of complement activation and macrophage chemotaxis in AMD, molecular mechanisms of choroidal neovascularization, and the roles of oxidative damage and lipid metabolism. Finally, the article gives an overview of spontaneous and induced nonhuman primate models and describes relevant mouse models in the context of each pathogenetic mechanism.
Voluntary movement in animals is modulated by a number of subcortical systems. One of these resides in the basal nuclei and their associated projections and utilizes dopamine as a neurotransmitter. ...Apart from regulating movement, the dopaminergic axis is also involved in the control of goal-oriented behavior, cognition, and mood. Disorders of this system result in common human neurologic disorders such as Parkinson's and Huntington's diseases, as well contributing to a host of behavioral conditions, such as schizophrenia, attention deficit hyperactivity disorder, and addiction. Many individual mouse models of human dopaminergic dysfunction have been described in varying degrees of detail. However, when evaluating this region of the brain, the veterinary pathologist is confronted by a paucity of information summarizing the comparative aspects of the anatomy, physiology, and pathology of the central dopaminergic system. In this review, a systematic approach to anatomic phenotyping of the central dopaminergic system in the mouse is described and illustrated using tyrosine hydroxylase immunohistochemistry. Differences between murine neuroanatomy and comparable regions of the nonhuman primate brain are highlighted. Although the mouse is the focus of this review, conditions in domestic animals characterized by lesions within the basal nuclei and its projections are also briefly described. Murine behavioral and motor tests that accompany abnormalities of specific anatomic regions of the dopaminergic axis are summarized. Finally, we review mouse models of Parkinson's and Huntington's diseases, as well as those genetically altered mice that elucidate aspects of dopamine metabolism and receptor function.
A phenotyping study records physiologic or morphologic changes in an experimental animal resulting from an intervention. In mice, this intervention is most frequently genetic, but it may be any type ...of experimental manipulation. Accurate representation of the human condition under study is essential if the model is to yield useful conclusions. In this review, general approaches to the design of phenotyping studies are considered. These approaches take into account major sources of reduced model validity, such as unexpected phenotypic variation in mice, evolutionary divergence between mice and humans, unanticipated sources of variation, and common design errors. As poor design is the most common reason why studies fail to yield enduring results, emphasis is placed on reduction of bias, sampling, controlled study design, and appropriate statistical analysis.
The waved with open eyes (woe) locus is a spontaneous recessive mouse mutation that exhibits wavy fur, eyelids open at birth, and enlarged heart and esophagus. In this study, we confirmed the ...previously identified woe phenotypes and additionally identified anterior eye segment defects, absence of the meibomian glands, and defects in the semilunar cardiac valves. Positional cloning identified a C794T substitution in the Adam17 gene that ablates a putative exonic splicing enhancer (ESE) sequence in exon 7 resulting in aberrant Adam17 splicing. The predominant woe transcript, Adam17(Delta)(exon7), lacks exon 7 resulting in an in-frame deletion of 90 bp and a putative Adam17(Delta252-281) protein lacking residues 252-281 from the metalloprotease domain. Western blot analysis in woe identified only the precursor form of Adam17(Delta252-281) protein. Absence of cleavage of the prodomain renders Adam17(Delta252-281) functionally inactive; however, constitutive and stimulated shedding of Adam17 substrates was detected in woe at significantly reduced levels. This residual Adam17 shedding activity in woe most likely originates from full-length Adam17(T265M) encoded by the Adam17(C794T) transcript identified expressed at severely reduced levels. These results show that even small amounts of functional Adam17 allow woe mice to survive into adulthood. In contrast to Adam17(-/-) mice that die at birth, the viability of woe mice provides an excellent opportunity for studying the role of Adam17 throughout postnatal development and homeostasis.
Feline ocular sarcomas are malignant intraocular neoplasms that are frequently associated with a history of ocular trauma. They usually present as fibrosarcomas, but some have both epithelial and ...mesenchymal features. The purpose of this study was to determine the cell of origin of a subset of feline intraocular sarcomas that display a mixed epithelial-mesenchymal phenotype, with elaboration of basement membrane—type matrix. We examined the morphology and histochemical and immunohistochemical phenotypes of nine feline intraocular sarcomas. Immunohistochemistry and in situ hybridization were performed to detect expression of crystallin alpha A. In addition, tumors were examined for expression of vimentin, cytokeratin, smooth muscle actin, desmin, melan A, neural cell adhesion molecule, S-100, glial fibrillary acidic protein, nerve growth factor receptor, and collagen type IV. Animals ranged from 7 to 17 years of age—no breed or sex predilection for tumor occurrence was present. Tumors were characterized by mixed epithelial and mesenchymal phenotypes, both of which elaborated basement membrane-type material and expressed vimentin highly. On the basis of collagen type IV and crystallin alpha A immunopositivity, we established that three of nine tumors were of lens epithelial origin. Expression of desmin and smooth muscle actin identified one tumor as a leiomyosarcoma. The remainder were undifferentiated sarcomas of myofibroblastic origin. This is the first report of lens epithelial neoplasia in clinical material from any species. The history and morphologic features of feline ocular sarcomas are reminiscent of feline vaccine-induced sarcomas. These tumors may share pathophysiologic similarities unique to this species.
Abstract Background Elucidating the functional and structural alterations in skeletal muscle perfusion and perfusion reserve is pivotal for the effective management of peripheral artery disease ...(PAD). This investigation correlates functional imaging metrics with structural histological modifications in a rabbit hindlimb ischemia model (HLI). Methods HLI was created in rabbits (n=8) by right femoral ligations and Rb-82 PET/CT imaging performed on digital PET scanner (Vision600, Siemens) at either 2 or 4 weeks post-ligation. Rabbits were injected with Rb-82 (1.5 mCi) with constant activity infusion over 30 sec at rest and 30 sec following reactive hyperemia (RH) induced by 3 min cuff occlusion of thighs bilaterally. PET images were acquired with shuttling scanner from heart to feet using 8 passes over 7 min. Summed images were created excluding the first 90 sec post injection. Calf muscles were segmented based on CT images. Mean Rb-82 standardized uptake values (SUVs) were calculated for calf muscles - gastrocnemius (GC), soleus (SOL) for both ischemic (IS) and non-ischemic (NI) limbs. CT angiography was performed, and tissues analyzed for fiber composition using Fast myosin (FM) that stains type 2 muscle fibers, collagen area percentages as an indicator of fibrosis using Mason trichrome (MT), and capillary densities using CD31 in the two distinct classical oxidative Type 1 (SOL) and glycolytic Type 2 (GC) muscles. Results PET imaging demonstrated differential perfusion and perfusion reserve among calf muscles (Fig. 1A, 1B). While no marked difference was observed between GC or SOL (IS vs. NI) in either rest or stress conditions, the SOL exhibited a higher Rb-82 uptake than GC during both conditions (P<0.05). IS to NI uptake ratios during RH were lower than at rest, suggesting RH may identify subtle perfusion deficits in IS limbs (P<0.05). Histologically, both GC and SOL showed an increase in capillary densities in IS limb (GC: NI 1.8 ± 0.3, IS 2.2 ± 0.2 , p=0.01; SOL: NI 2.7 ± 0.2, IS 3.1 ± 0.4 , p=0.03), while SOL had a significantly higher capillary density compared to GC (p<0.05). FM staining revealed consistent Type 2 myofiber prevalence in GC across IS and NI conditions (NI 84.6% ± 10.2%, IS 85.0% ± 9.3%, ns) with minimal Type 2 staining in SOL (NI 2.1% ± 4.1%, IS 1.9% ± 5.2%, ns); SOL vs GC (P<0.01). MT staining indicated stable collagen levels (GC NI 1.2% ± 0.6%, IS 1.3% ± 1.0%, ns; SOL: NI 2.7% ± 1.0%, IS 2.4% ± 0.7%, ns) (Fig. 1 C-E). Conclusion PET imaging of the lower extremities demonstrated increased perfusion in SOL versus GC in association with an increased capillary to muscle fiber ratio. A reduction in IS/NI perfusion ratio was not detected at rest although could be detected during RH. The integration of histological findings with skeletal muscle perfusion provides useful pathophysiological insight in our clinical relevant pre-clinical model of PAD, offering a valuable approach to evaluate treatment strategies in PAD.
To establish whether photoreceptor apoptosis in the rd-1 mouse is accompanied by cell cycle progression. Studies of cell cycle proteins in other models of neuronal death provide consistent evidence ...that a repertoire of proliferative markers accompanies apoptosis.
The spatiotemporal progression of photoreceptor loss in rd-1 and control mice at postnatal days (PN)8, -10, -12, -15, and -18 was correlated with markers of G(1)- and S-phase progression. Photoreceptor death was detected by using morphology and terminal dUTP transferase nick end labeling (TUNEL). Cell-cycle-associated markers consisted of bromodeoxyuridine (BrdU) uptake, and immunolabeling for proliferating cell nuclear antigen (PCNA), Ki-67, and cyclin-dependent kinases-2 and -4. The identity of proliferating cells in the outer nuclear layer was established by double immunolabeling with PCNA and either F4/80 or recoverin.
A population of proliferating cells in the outer nuclear layer accompanies photoreceptor death along a central to peripheral gradient in rd-1 retinas. Double immunolabeling for PCNA and F4/80 readily identified these as microglial cells originating from the inner retina. Cell cycle progression in photoreceptors could not be demonstrated.
These findings confirm that in rd-1, a preexisting condition for cell cycle progression does not exist as it does in other neurodegenerative conditions. Therefore, in this model, evidence of photoreceptor cell cycle progression in retinas exposed to neurotrophic factors is likely to result from the therapy itself. In addition, the results confirmed that proliferating microglial cells are intimately associated with the degenerative process in rd-1.
Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by ...subretinal delivery of AAV-RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2.RPE65 in RPE65-mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vectorrelated toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2.RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2.RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina.
X-linked progressive retinal atrophy (XLPRA) in the Siberian husky dog is a naturally occurring X-linked retinopathy closely resembling X-linked retinitis pigmentosa (XLRP) in humans. In affected ...males, initial degeneration of rods is followed by cone degeneration and complete retinal atrophy; carrier females have random patches of rod degeneration consistent with random X chromosome inactivation. By typing the XLPRA pedigree with five intragenic markers dystrophin, retinitis pigmentosa GTPase regulator ( RPGR ), tissue inhibitor of metalloproteinases 1, androgen receptor and factor IX, we established a linkage map of the canine X chromosome, and confirmed that the order of these five genes is identical to that on the human X. XLPRA was tightly linked to an intragenic RPGR polymorphism (LOD 11.7, zero recombination), thus confirming locus homology with RP3. We cloned the full-length canine RPGR cDNA and three additional splice variants. No disease-causing mutation was found in the RPGR-coding sequence of the four splice variants characterized, a finding similar to approximately 80% of human XLRP patients whose disease maps to the RP3 locus. In addition, there were no significant differences in the proportional expression of each splice variant in normal and pre-degenerate XLPRA-affected retina. Expression of all RPGR splice variants increased later in the disease, when retinas were undergoing active degeneration. The results provide further evidence of cross-species retention of a complex splicing pattern in the 3' portion of RPGR, the functional significance of which is unknown. In addition, the possibility of another disease locus in the RP3 region is supported.
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