Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells.

Tree shrews ( Tupaia belangeri chinensis) share a close relationship to primates and have been widely used in biomedical research. We previously established a spermatogonial stem cell (SSC)-based gene editing platform to generate transgenic tree shrews. However, the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear. Here, we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages. We found that SSCs lost spermatogenesis ability after long-term expansion (>50 passages), as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia (SPG)-derived spermatocytes or spermatids marking spermatogenesis. RNA sequencing (RNA-seq) analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers. Specifically, DNA damage response and repair genes (e.g., MRE11, SMC3, BLM, and GEN1) were down-regulated, whereas genes associated with mitochondrial function (e.g., NDUFA9, NDUFA8, NDUFA13, and NDUFB8) were up-regulated after expansion. The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells. Supplementation with nicotinamide adenine dinucleotide (NAD +) precursor nicotinamide riboside (NR) exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture. Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance.

A comparison of behavioral paradigms assessing spatial memory in tree shrews.

Impairments in spatial navigation in humans can be preclinical signs of Alzheimer's disease. Therefore, cognitive tests that monitor deficits in spatial memory play a crucial role in evaluating animal models with early stage Alzheimer's disease. While Chinese tree shrews (Tupaia belangeri) possess many features suitable for Alzheimer's disease modeling, behavioral tests for assessing spatial cognition in this species are lacking. Here, we established reward-based paradigms using the radial-arm maze and cheeseboard maze for tree shrews, and tested spatial memory in a group of 12 adult males in both tasks, along with a control water maze test, before and after bilateral lesions to the hippocampus, the brain region essential for spatial navigation. Tree shrews memorized target positions during training, and task performance improved gradually until reaching a plateau in all 3 mazes. However, spatial learning was compromised post-lesion in the 2 newly developed tasks, whereas memory retrieval was impaired in the water maze task. These results indicate that the cheeseboard task effectively detects impairments in spatial memory and holds potential for monitoring progressive cognitive decline in aged or genetically modified tree shrews that develop Alzheimer's disease-like symptoms. This study may facilitate the utilization of tree shrew models in Alzheimer's disease research.

What and Where: Location-Dependent Feature Sensitivity as a Canonical Organizing Principle of the Visual System.

Traditionally, functional representations in early visual areas are conceived as retinotopic maps preserving ego-centric spatial location information while ensuring that other stimulus features are uniformly represented for all locations in space. Recent results challenge this framework of relatively independent encoding of location and features in the early visual system, emphasizing location-dependent feature sensitivities that reflect specialization of cortical circuits for different locations in visual space. Here we review the evidence for such location-specific encoding including: (1) systematic variation of functional properties within conventional retinotopic maps in the cortex; (2) novel periodic retinotopic transforms that dramatically illustrate the tight linkage of feature sensitivity, spatial location, and cortical circuitry; and (3) retinotopic biases in cortical areas, and groups of areas, that have been defined by their functional specializations. We propose that location-dependent feature sensitivity is a fundamental organizing principle of the visual system that achieves efficient representation of positional regularities in visual experience, and reflects the evolutionary selection of sensory and motor circuits to optimally represent behaviorally relevant information. Future studies are necessary to discover mechanisms underlying joint encoding of location and functional information, how this relates to behavior, emerges during development, and varies across species.

Mechanism underpinning the sharpening of orientation and spatial frequency selectivities in the tree shrew (Tupaia belangeri) primary visual cortex.

Most neurons in the primary visual cortex (V1) of mammals show sharp orientation selectivity and band-pass spatial frequency tuning. Here, we examine whether sharpening of the broad tuning that exists subcortically, namely in the retina and the lateral geniculate nucleus (LGN), underlie the sharper tuning seen for both the above features in tree shrew V1. Since the transition from poor feature selectivity to sharp tuning occurs entirely within V1 in tree shrews, we examined the orientation selectivity and spatial frequency tuning of neurons within individual electrode penetrations. We found that most layer 4 and layer 2/3 neurons in the same cortical column preferred the same stimulus orientation. However, a subset of layer 3c neurons close to the layer 4 border preferred near orthogonal orientations, suggesting that layer 2/3 neurons may inherit the orientation preferences of their layer 4 input neurons and also receive cross-orientation inhibition from layer 3c neurons. We also found that layer 4 neurons showed sharper orientation selectivity at higher spatial frequencies, suggesting that attenuation of low spatial frequency responses by spatially broad inhibition acting on layer 4 inputs to layer 2/3 neurons can enhance both orientation and spatial frequency selectivities. However, in a proportion of layer 2/3 neurons, the sharper tuning of layer 2/3 neurons appeared to arise also or even mainly from inhibition specific to high spatial frequencies acting on the layer 4 inputs to layer 2/3. Overall, our results are consistent with the suggestion that in tree shrews, sharp feature selectivity in layer 2/3 can be established by intracortical mechanisms that sharpen biases observed in layer 4, which are in turn inherited presumably from thalamic afferents.

Animal Models in Human Adenovirus Research.

Human adenovirus (HAdV) infections cause a wide variety of clinical symptoms, ranging from mild upper respiratory tract disease to lethal outcomes, particularly in immunocompromised individuals. To date, neither widely available vaccines nor approved antiadenoviral compounds are available to efficiently deal with HAdV infections. Thus, there is a need to thoroughly understand HAdV-induced disease, and for the development and preclinical evaluation of HAdV therapeutics and/or vaccines, and consequently for suitable standardizable in vitro systems and animal models. Current animal models to study HAdV pathogenesis, persistence, and tumorigenesis include rodents such as Syrian hamsters, mice, and cotton rats, as well as rabbits. In addition, a few recent studies on other species, such as pigs and tree shrews, reported promising data. These models mimic (aspects of) HAdV-induced pathological changes in humans and, although they are relevant, an ideal HAdV animal model has yet to be developed. This review summarizes the available animal models of HAdV infection with comprehensive descriptions of virus-induced pathogenesis in different animal species. We also elaborate on rodent HAdV animal models and how they contributed to insights into adenovirus-induced cell transformation and cancer.

Establishment of Tree Shrew Animal Model for Kaposi's Sarcoma-Associated Herpesvirus (HHV-8) Infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the most common cause of Kaposi's sarcoma (KS) and other malignant growths in humans. However, the lack of a KSHV-infected small animal model has hampered understanding of the mechanisms of KSHV infection, virus replication, pathogenesis, and persistence. This study was designed to explore the susceptibility of tree shrews as a possible KSHV-infected small animal model. A recombinant GFP (latent)/RFP (lytic)-positive rKSHV.219 strain was used to infect primary cells cultured from different tissues of tree shrews as an in vitro model and adult tree shrews as an in vivo model. KSHV latent nuclear antigen (LANA) and DNA were successfully detected in primary cells of tree shrews. Among them, tree shrew kidney epithelial cells (TSKEC) were the most susceptible cells to KSHV infection compared to other cells. KSHV genomic DNA, mRNA, and KSHV-specific proteins were readily detected in the TSKEC cultured up to 32 dpi. Moreover, KSHV DNA and mRNA transcription were also readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of tree shrews infected with KSHV. Haematoxylin and eosin (HE) staining showed lymphocyte infiltration, lymphoid tissue focal aggregation, alveolar wall thickening, hepatocyte edema, hepatic necrosis in the spleen, lung, and liver of KSHV-infected animals. Additionally, immune-histochemical (IHC) staining showed that LANA or ORF62-positive cells were present in the spleen, lung, liver, and kidney of KSHV-infected tree shrews. Here, we have successfully established in vitro and in vivo KSHV latent infection in tree shrews. This small animal model is not only useful for studying the pathogenesis of KSHV in vivo but can also be a useful model to study transmission routes of viral infection and a useful platform to characterize the novel therapeutics against KSHV.

Characterization of tree shrew telomeres and telomerase.

The use of tree shrews as experimental animals for biomedical research is a new practice. Several recent studies suggest that tree shrews are suitable for studying cancers, including breast cancer, glioblastoma, lung cancer, and hepatocellular carcinoma. However, the telomeres and the telomerase of tree shrews have not been studied to date. Here, we characterize telomeres and telomerase in tree shrews. The telomere length of tree shrews is approximately 23 kb, which is longer than that of primates and shorter than that of mice, and it is extended in breast tumor tissues according to Southern blot and flow-fluorescence in situ hybridization (FISH) analyses. Tree shrew spleen, bone marrow, testis, ovary, and uterus show high telomerase activities, which are increased in breast tumor tissues by telomeric repeat amplification protocol assays. The telomere length becomes shorter, and telomerase activity decreases with age. The tree shrew TERT and TERC are more highly similar to primates than to rodents. These findings lay a solid foundation for using tree shrews to study aging and cancers.

Tree Shrew Cells Transduced with Human CD4 and CCR5 Support Early Steps of HIV-1 Replication, but Viral Infectivity Is Restricted by APOBEC3.

The host range of human immunodeficiency virus type 1 (HIV-1) is narrow. Therefore, using ordinary animal models to study HIV-1 replication, pathogenesis, and therapy is impractical. The lack of applicable animal models for HIV-1 research spurred our investigation on whether tree shrews (Tupaia belangeri chinensis), which are susceptible to many types of human viruses, can act as an animal model for HIV-1. Here, we report that tree shrew primary cells are refractory to wild-type HIV-1 but support the early replication steps of HIV-1 pseudotyped with the vesicular stomatitis virus glycoprotein envelope (VSV-G), which can bypass entry receptors. The exogenous expression of human CD4 renders the tree shrew cell line infectible to X4-tropic HIV-1IIIB, suggesting that tree shrew CXCR4 is a functional HIV-1 coreceptor. However, tree shrew cells did not produce infectious HIV-1 progeny virions, even with the human CD4 receptor. Subsequently, we identified tree shrew (ts) apolipoprotein B editing catalytic polypeptide 3 (tsAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity, with virus infectivity reduced 10- to 1,000-fold. Unlike human APOBEC3G, the tsA3Z2c-Z1b protein was not degraded by the HIV-1 viral infectivity factor (Vif) but markedly restricted HIV-1 replication through mutagenicity and reverse transcription inhibition. The pooled knockout of tsA3Z2c-Z1b partially restored the infectivity of the HIV-1 progeny. This work suggests that tsAPOBEC3 proteins serve as an additional barrier to the development of HIV-1 tree shrew models, even when virus entry is overcome by exogenous expression of human CD4. IMPORTANCE The development of animal models is critical for studying human diseases and their pathogenesis and for evaluating drug and vaccine efficacy. For improved AIDS research, the ideal animal model of HIV-1 infection should be a small laboratory mammal that closely mimics virus replication in humans. Tree shrews exhibit considerable potential as animal models for the study of human diseases and therapeutic responses. Here, we report that human CD4-expressing tree shrew cells support the early steps of HIV-1 replication and that tree shrew CXCR4 is a functional coreceptor of HIV-1. However, tree shrew cells harbor additional restrictions that lead to the production of HIV-1 virions with low infectivity. Thus, the tsAPOBEC3 proteins are partial barriers to developing tree shrews as an HIV-1 model. Our results provide insight into the genetic basis of HIV inhibition in tree shrews and build a foundation for the establishment of gene-edited tree shrew HIV-1-infected models.

Increased cocaine motivation in tree shrews is modulated by striatal dopamine D1 receptor-mediated upregulation of Cav 1.2.

The progressively increased motivation for cocaine during abstinence is closely associated with the dysfunction of dopamine (DA) system. As DA receptors also dynamically regulate L-type calcium channels (LTCCs), in this study we examined how DA receptors (D1R or D2R) and LTCCs (Cav 1.2 or Cav 1.3) exert their influences on cocaine-seeking in a tree shrew (Tupaia belangeri chinensis) model. First, we demonstrated the 'incubation' effect by showing tree shrews exhibited a significantly higher seeking behaviour on withdrawal day (WD) 45 than on WD1. Then, we confirmed that longer abstinence period induced higher D1R expression in the nucleus accumbens (NAc). Next, we showed that LTCCs in the NAc participated in drug seeking. Moreover, Cav 1.2 expression in the NAc was increased on WD45, and disruption of the Cav 1.2 inhibited drug seeking. Finally, we found that D1R antagonist blocked the increase of Cav 1.2 on drug-seeking test. Collectively, these findings suggest that D1R-mediated upregulation of Cav 1.2 is involved in the incubation of cocaine craving.

cDNA cloning, expression and bioinformatical analysis of Tssk genes in tree shrews.

Tree shrews are more closely related to primate animals than rodents in many aspects. In addition, they also possess several advantageous characteristics including small body size, high brain-to-body mass ratio, low cost of feeding and maintenance, short reproductive cycle and life span, which make them promising novel laboratory animals to replace more precious larger primate animals. Testis-specific serine/threonine kinase (Tssk) plays important roles in spermatogenesis and/or the regulation of sperm function. However, studies on Tssk in tree shrews have not been reported yet. In the present study, the full-length sequences of five members of the Tssk family in tree shrews were cloned and their CDS region sequences were analyzed by basic bioinformatics. The phylogenetic tree and prokaryotic protein expression system of Tssk gene of tree shrews were constructed. The mRNA expressions of Tssk genes in 11 tissues/organs from tree shrews were studied. The results showed that: 1. the length of the CDS region of tree shrew Tssk gene for Tssk1B, Tssk2, Tssk3 (variant X1 / X2), Tssk4 (variant X1 / X2) and Tssk6 is 1080bp, 1077bp, 867 / 807bp, 1014 / 984bp, 822bp, respectively, encoding 359, 358, 288/268, 337/327 and 273 amino acids, respectively; the cloned sequences of Tssk genes have been submitted to GenBank with the following accession numbers: KX091161(Tssk1B), KX091162(Tssk2), KX091163(Tssk3 variant X1)/KX091164(Tssk3 variant X2), KX091165(Tssk4variant X1)/KX091166(Tssk4variant X2), KX091160(Tssk6). 2. All tree shrew Tssk proteins distribute in cytoplasm, indicating that they are hydrophilic and non-secretory proteins, with multiple phosphorylation sites of serine and/or threonine. In addition, they are all mixed proteins with similar tertiary structures sharing a highly conserved functional domain of S_TKc (Serine/Threonine protein kinases, catalytic domain). 3.The molecular phylogenetic tree of five Tssk genes in tree shrews indicates that they are neither rodent nor primate animal, but are closely related to primate animals. 4. Five members of the Tssk recombinant proteins in tree shrews were successfully obtained using the constructed prokaryotic protein expression system. 5. Five Tssk genes are specifically expressed in the testis and/or sperm of tree shrews. Additionally, small amount of Tssk1B was expressed in several tissues other than testis and sperm. Limited mRNA levels of Tssk2 and Tssk4 were expressed in the brain, while mRNA of Tssk3 or Tssk6 could only be detected in the testis and sperm. This study will provide fundamental data on reproductive biology of tree shrews, which paves a way for further studying Tssk's biological function in this novel model animal.

A Novel Tree Shrew Model of Diabetic Retinopathy.

Existing animal models with rod-dominant retinas have shown that hyperglycemia injures neurons, but it is not yet clearly understood how blue cone photoreceptors and retinal ganglion cells (RGCs) deteriorate in patients because of compromised insulin tolerance. In contrast, northern tree shrews (Tupaia Belangeri), one of the closest living relatives of primates, have a cone-dominant retina with short wave sensitivity (SWS) and long wave sensitivity (LWS) cones. Therefore, we injected animals with a single streptozotocin dose (175 mg/kg i.p.) to investigate whether sustained hyperglycemia models the features of human diabetic retinopathy (DR). We used the photopic electroretinogram (ERG) to measure the amplitudes of A and B waves and the photopic negative responses (PhNR) to evaluate cone and RGC function. Retinal flat mounts were prepared for immunohistochemical analysis to count the numbers of neurons with antibodies against cone opsins and RGC specific BRN3a proteins. The levels of the proteins TRIB3, ISR-1, and p-AKT/p-mTOR were measured with western blot. The results demonstrated that tree shrews manifested sustained hyperglycemia leading to a slight but significant loss of SWS cones (12%) and RGCs (20%) 16 weeks after streptozotocin injection. The loss of BRN3a-positive RGCs was also reflected by a 30% decline in BRN3a protein expression. These were accompanied by reduced ERG amplitudes and PhNRs. Importantly, the diabetic retinas demonstrated increased expression of TRIB3 and level of p-AKT/p-mTOR axis but reduced level of IRS-1 protein. Therefore, a new non-primate model of DR with SWS cone and RGC dysfunction lays the foundation to better understand retinal pathophysiology at the molecular level and opens an avenue for improving the research on the treatment of human eye diseases.

A tree shrew model for steroid-associated osteonecrosis.

Osteonecrosis is a common human disease in orthopedics. It is difficult to treat, and half of patients may need artificial joint replacement, resulting in a considerable economic burden and a reduction in quality of life. Hormones are one of the major causes of osteonecrosis and high doses of corticosteroids are considered the most dangerous factor. Because of the complexity of treatment, we still need a better animal model that can be widely used in drug development and testing. Tree shrews are more closely related to primates than rodents. As such, we constructed a successful tree shrew model to establish and evaluate steroid-associated osteonecrosis (SAON). We found that low-dose lipopolysaccharide (LPS) combined with high-dose methylprednisolone (MPS) over 12 weeks could be used to establish a tree shrew model with femoral head necrosis. Serum biochemical and histological analyses showed that an ideal model was obtained. Thus, this work provides a useful animal model for the study of SAON and for the optimization of treatment methods.

COVID-19-like symptoms observed in Chinese tree shrews infected with SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to pose a global threat to the human population. Identifying animal species susceptible to infection with the SARS-CoV-2/ HCoV-19 pathogen is essential for controlling the outbreak and for testing valid prophylactics or therapeutics based on animal model studies. Here, different aged Chinese tree shrews (adult group, 1 year old; old group, 5-6 years old), which are close relatives to primates, were infected with SARS-CoV-2. X-ray, viral shedding, laboratory, and histological analyses were performed on different days post-inoculation (dpi). Results showed that Chinese tree shrews could be infected by SARS-CoV-2. Lung infiltrates were visible in X-ray radiographs in most infected animals. Viral RNA was consistently detected in lung tissues from infected animals at 3, 5, and 7 dpi, along with alterations in related parameters from routine blood tests and serum biochemistry, including increased levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN). Histological analysis of lung tissues from animals at 3 dpi (adult group) and 7 dpi (old group) showed thickened alveolar septa and interstitial hemorrhage. Several differences were found between the two different aged groups in regard to viral shedding peak. Our results indicate that Chinese tree shrews have the potential to be used as animal models for SARS-CoV-2 infection.

Involvement of dopamine D3 receptor and dopamine transporter in methamphetamine-induced behavioral sensitization in tree shrews.

INTRODUCTION: This study aims to establish a methamphetamine (METH)-induced behavioral sensitization model using tree shrews, as well as to measure the protein expression of the dopamine D3 receptor (D3R) and dopamine transporter (DAT). METHODS: Forty tree shrews were equally and randomly divided into four experimental groups: those administered with 1, 2, and 4 mg/kg METH and a control group (treated with an equal amount of normal saline). Each experimental group was repeatedly exposed to METH for nine consecutive days to induce the development of behavioral sensitization, followed by four days of withdrawal (without the METH treatment) to induce the transfer of behavioral sensitization, then given 0.5 mg/kg of METH to undergo the expression of behavioral sensitization. Altered locomotor and stereotypic behaviors were measured daily via open-field experiments during the development and expression stages, and weight changes were also recorded. Then, the Western blot method was used to detect the expression levels of D3R and DAT in three brain regions: the nucleus accumbens, prefrontal cortex, and dorsal striatum 24 hr after the last behavioral test. RESULTS: METH administration augmented motor-stimulant responses and stereotypic behaviors in all experimental groups, and stereotypic behaviors intensified more in the groups treated with 2 and 4 mg/kg METH. Motion distance, speed, and trajectory were significantly elevated in all experimental, however, METH at 4 mg/kg induced more stereotypic behaviors, decreasing these locomotor activities as compared with the 2 mg/kg METH group. 2 and 4 mg/kg METH significantly upregulated and downregulated D3R and DAT expression levels, respectively, in three brain regions, and these changes are more pronounced in 2 mg/kg METH. CONCLUSIONS: These results indicated that this animal model may be used to study the neurobiological mechanisms that underly the development and expression of behavioral sensitization to METH. Deregulated D3R and DAT expression may be involved in the METH-induced behavioral sensitization.

Radiation-induced impairment of optic nerve axonal transport in tree shrews and rats monitored by longitudinal manganese-enhanced MRI.

PURPOSE: Radiation-induced optic neuropathy (RION) is a serious complication that occurs after radiation therapy of tumors in the vicinity of the optic nerve, yet its mechanism and imaging features are poorly understood. In this study, we employed manganese-enhanced MRI (MEMRI) to assess optic nerve axonal transport in tree shrews and rats after irradiation. MATERIALS AND METHODS: A comparison of normal visual projections in tree shrews and rats was conducted by intravitreal MnCl2 injection followed by MRI. Adult male tree shrews and rats received a total dose of 20 Gy delivered in two fractions (10 Gy per fraction) within 5 days. Longitudinal MEMRI was conducted 5, 10, 20 and 30 weeks after radiation. At the end of observation, motor proteins involved in axonal transport were detected by western blotting, and the axon cytoskeleton was assessed by immunofluorescence. RESULTS: The eyeballs, lens sizes, vitreous volumes, optic nerves and superior colliculi of tree shrews were significantly larger than those of rats on MEMRI (P < 0.05). The Mn2+-enhancement of the optic nerve showed no significant changes at 5 and 10 weeks (P > 0.05) but decreased gradually from 20 to 30 weeks postirradiation (P < 0.05). The enhancement of the superior colliculus gradually decreased from 5 weeks to 30 weeks, and the decrease was most significant at 30 weeks (P < 0.05). The levels of the motor proteins cytoplasmic dynein-1, kinesin-1 and kinesin-2 in the experimental group were significantly decreased (P < 0.05). The immunofluorescence results showed that the α-tubulin, ß-tubulin and SMI 31 levels in the experimental groups and control groups were not significantly different (P > 0.05). CONCLUSION: Tree shrews show great advantages in visual neuroscience research involving MEMRI. The main cause of the decline in axonal transport in RION is an insufficient level of motor protein rather than damage to the axonal cytoskeletal structure. Longitudinal MEMRI can be used to detect changes in axonal transport function and to observe the relatively intact axon structure from the early to late stages after radiation administration.

Chinese tree shrews as a primate experimental animal eligible for the study of alcoholic liver disease: characterization and confirmation by MRI.

There has been a lack of suitable fatty liver models and characterization techniques for histopathological evaluation of alcoholic fatty liver (AFL). This work aimed to exploit an magnetic resonance imaging (MRI) technique for characterizing an alcohol-induced fatty liver model established in tree shrews (Tupaia belangeri chinese). The animals were treated with 15% alcohol for two weeks instead of drinking water to induce AFL. Blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), alcohol, and liver malondialdehyde (MDA) concentrations were determined, and the histopathology of the liver was checked by hematoxylin & eosin (HE) and Oil red O staining on day 0 and on the 4th, 7th and 14th days after alcohol feeding. MRI was used to trace the histopathological changes in the liver of tree shrews in real time. Compared with the control group, the levels of ALT, AST, and MDA significantly increased in the alcohol-induced group and were positively correlated with the induction time. HE and Oil red O staining revealed that a moderate fatty lesion occurred in the liver on the 4th day and that a serious AFL was successfully induced on the 14th day. MRI further confirmed the formation of AFL. MRI, as noninvasive examination technique, provides an alternative tool for accurate characterization of AFL in live subjects. It is comparable to HE or Oil red O staining for histopathological examination, but is more suitable by virtue of its high flexibility and compliance. The AFL model of tree shrews combined with MRI characterization can work as a platform for studying fatty liver diseases and medications for their treatment.

Detection and characterization of tree shrew retinal venous pulsations: An animal model to study human retinal venous pulsations.

Spontaneous retinal venous pulsations (SRVPs), pulsations of branches of the central retinal vein, are affected by intraocular pressure (IOP) and intracranial pressure (ICP) and thus convey potentially-useful information about ICP. However, the exact relationship between SRVPs, IOP, and ICP is unknown. It is not easily feasible to study this relationship in humans, necessitating the use of an animal model. We here propose tree shrews as a suitable animal model to study the complex relationship between SRVPs, IOP, and ICP. Tree shrew SRVP incidence was determined in a population of animals. Following validation of a modified IOP control system to accurately and quickly control IOP, IOP and/or ICP were manipulated in two tree shrews with SRVPs and the effects on SRVP properties were quantified. SRVPs were present in 75% of tree shrews at physiologic IOP and ICP. Altering IOP or ICP produced changes in tree shrew SRVP properties; specifically, increasing IOP caused SRVP amplitude to increase, while increasing ICP caused SRVP amplitude to decrease. In addition, a higher IOP was necessary to generate SRVPs at a higher ICP than at a lower ICP. SRVPs occur with a similar incidence in tree shrews as in humans, and tree shrew SRVPs are affected by changes in IOP and ICP in a manner qualitatively similar to that reported in humans. In view of anatomic similarities, tree shrews are a promising animal model system to further study the complex relationship between SRVPs, IOP, and ICP.

Establishment of brain ischemia model in tree shrew.

BACKGROUND: Tree shrew, as a kind of small and inexpensive animal between insectivores and primates with the general anatomy being similar to human, could be considered as developed animal model for brain ischemia (BI) study. However, there is no neural behavior scores criterion from tree shrew with BI up to now. METHODS: To produce BI model of tree shrew, a novel systematic neurobehavioral assessment scale, named as neural behavior scores (NBS) including aggressive behavior, seeking behavior, gait, startle reflex, high jump and warped-tail phenomenon was firstly established and used in this study. Moreover, magnetic resonance imaging (MRI) was performed on the first day after the operation to detect the imaging changes caused by ischemia. Then TTC, HE staining and immunofluorescent staining for GFAP and NeuN, were performed 24 h after surgery respectively. RESULTS: NBS in BI group were significantly higher than that of sham operation group at 1d, 3d, 5d and 7d after ischemia. Meanwhile, compared with the sham operation group, the T2 images demonstrated significant higher signal and local brain swelling after cerebral ischemia in tree shrews. The staining of TTC and HE showed apparent infarction and necrosis of the cerebral region, and most of neurons exhibited a shrink. CONCLUSION: We have successfully established the BI model of tree shrew, confirmed by NBS (a new developed method), MRI, HE staining, TTC staining and immunofluorescence staining. It is the first time to report a novel neurobehavioral assessment scale for BI in tree shrew.

Establishment of an immortalized intestinal epithelial cell line from tree shrews by lentivirus-mediated hTERT gene transduction.

The intestinal epithelium has an average lifespan of 4-5 days. Normally, primary intestinal epithelial cells can be cultured for about 15 days in vitro. The aim of this study was to explore methods to isolate and immortalize intestinal epithelial cells (IECs) of tree shrews in order to establish a new resource of experimental material and to provide a cell model for drug development and infection mechanism research. Tissue from the small intestine of tree shrews was digested with collagenase XI, neutral protease I, and dithiothreitol. The human telomerase reverse transcriptase gene (hTERT) was transferred into tree shrew IECs using the pHBLV-CMVIE-ZsGreen-Puro vector. The level of hTERT mRNA was detected by quantitative reverse transcription polymerase chain reaction. Immunofluorescence and western blot assays were performed to detect biochemical markers of IECs. The micromorphology of cells was observed with electron microscopy. We then conducted experiments to assess proliferative activity and analyze the karyotype of isolated cells. The results showed the immortalized cell line that we established and screened, maintained the characteristics and biochemical markers of primary IECs. Our results showed that the cell line we established can be considered an alternative cell model for intestinal drug research and for studies on intestinal infection and cell signaling.

Matching the LenStar optical biometer to A-Scan ultrasonography for use in small animal eyes with application to tree shrews.

We describe an analysis strategy to obtain ultrasonography-matched axial dimensions of small animal eyes using the LenStar biometer. The LenStar optical low-coherence reflectometer is an attractive device for animal research due to its high precision, non-invasiveness, and the ability to measure the axial dimensions of cornea, anterior chamber, lens, vitreous chamber, and axial length. However, this optical biometer was designed for clinical applications in human eyes and its internal analysis provides inaccurate values when used on small eyes due to species-dependent differences in refractive indices and relative axial dimensions. The LenStar uses a near infrared light source to measure optical path lengths (OPLs) that are converted by the LenStar's EyeSuite software into geometrical lengths (GLs) based on the refractive indices and axial dimensions of the human eye. We present a strategy that extracts the OPLs, determines refractive indices specific for the small animal eye of interest and then calculates corrected GLs. The refractive indices are obtained by matching the LenStar values to ultrasonography values in the same eyes. As compared to ultrasounography, we found that the internal calculations of the LenStar underestimate the axial dimensions of all ocular compartments of the tree shrew eye: anterior segment depth by 6.17±4.50%, lens thickness by 1.37±3.06%, vitreous chamber depth by 29.23±2.35%, and axial length by 10.62±1.75%. Using tree shrew-specific refractive indices, the axial dimensions closely matched those measured by ultrasonography for each compartment. Our analysis strategy can be easily translated to other species by obtaining a similar paired data set using ultrasonography and LenStar, and applying our step by step procedures.