Rowett Nude Research Articles

Development of an immunosuppressed orthotopic hepatocellular carcinoma rat model for the evaluation of chemo- and radioembolization therapies

Hepatocellular carcinoma (HCC) is widely known to be chemo-resistant and presents with significant liver disease resulting in low tolerability to systemic chemotherapy. As a counter measure, more targeted therapies such as trans-arterial chemoembolization (TACE) and trans-arterial radioembolization (TARE) have been developed. To further optimize these therapies, animal models are critical in elucidating the molecular events in disease progression and test new treatment options. The present study focuses on the development of a hepatoma bearing rat model. N1S1 rat hepatoma cells were transfected by a lentiviral method and injected into the liver of Sprague Dawley (SD) and Rowett Nude (RNU) rats. Longitudinal tumor growth was observed by bioluminescence imaging (BLI) and liver/tumor histology. In both models, tumors were visible within 4 days post cell inoculation. Tumor take rates were 52 % and 73 % for male and female SD rats, respectively, and 100 % for male RNU rats. By day 12 and 15 post inoculation, we recorded complete tumor regression in male and female SD rats. Liver histology showed advanced fibrosis in the tumor regressed SD rats, whilst RNU rats exhibited the characteristic sheet pattern of Novikoff tumor with mild liver fibrosis. Increased CD3 and TUNEL staining observed in SD rat livers may be key factors for tumor regression. Our data reveal that the immunocompetent SD rats are not recommended as a model for therapeutic investigations. The immunosuppressed RNU rats, however, are characterized by consistent and reliable tumor growth and thus a desirable model for future therapeutic investigations.

Targeting Myofibroblasts as a Treatment Modality for Dupuytren Disease

Currently, no treatment corrects the contractile nature of Dupuytren myofibroblasts (DMFs) or prevents recurrence following surgery. Antifibrotic and proadipogenic growth factors are released when adipose-derived stem cells (ASCs) are cultured with platelet-rich plasma (PRP), a platelet concentration from whole blood. Reprograming myofibroblasts into adipocytes via growth factors is proposed as a powerful potential tool to target fibrosis. We aimed to assess whether the combination of ASCs and PRP reprograms DMFs into adipocytes invitro and alters their contractile nature invivo. Normal human dermal fibroblasts (NHDFs) and DMFs from Dupuytren patients were isolated and cocultured with ASCs and PRP either alone or together. Adipocytes were detected by Oil Red O and perilipin staining. DMFs and NHDFs were transplanted into the forepaws of rats (Rowett Nude [rnu/rnu]) and treated with saline, PRP+ASCs, or collagenase Clostridium histolyticum (clinical comparison) 2 months later. After 2 weeks, the tissue was harvested and subjected to Masson trichrome staining, and collagen I and III and alpha-smooth muscle actin detection by immunohistochemistry. Myofibroblasts transform into adipocytes upon coculture with PRP+ASCs. DMFs show increased alpha-smooth muscle actin expression invivo compared with NHDFs, which is significantly decreased after PRP+ASCs and collagenase Clostridium histolyticum treatments. DMFs induce collagen I and III expressions in rat paws compared with NHDFs, with a type III to I ratio increase. Treatment with PRP+ASC reduced the ratio, but collagenase Clostridium histolyticum did not. Treating DMFs with PRP+ASCs provides factors that induce myofibroblast to adipocyte transformation. This treatment reduces the contractile phenotype and fibrosis markers invivo. Future studies should detail the mechanism of this conversion. The combination of PRP and ASCs to induce the differentiation of DMFs into adipocytes may serve to limit surgery to a percutaneous contracture release and biological injection, rather than a moderate or radical fasciectomy, and reduce the recurrence of Dupuytren contracture.

Intranasally applied human olfactory mucosa neural progenitor cells migrate to damaged brain regions.

Aim:To determine if intranasally administered olfactory mucosa progenitor cells (OMPCs) migrate to damaged areas of brain.Materials & methods:Rowett Nude (RNU) adult rats were injured using the Marmarou model then 2 weeks later received intranasally-delivered human OMPC. After 3 weeks, rats were sacrificed and brain sectioned. The mean distances from the human OMPCs to markers for degenerative neuronal cell bodies (p-c-Jun+), axonal swellings on damaged axons (β-APP+) and random points in immunostained sections were quantified. One-way ANOVA was used to analyze data.Results:The human OMPCs were seen in specific areas of the brain near degenerating cell bodies and damaged axons.Conclusion:Intranasally delivered human OMPC selectively migrate to brain injury sites suggesting a possible noninvasive stem cell delivery for brain injury.

Tissue Engineering of Lymphatic Vasculature in the Arteriovenous Loop Model of the Rat.

Various therapeutic approaches, for example, in case of trauma or cancer require the transplantation of autologous tissue. Depending on the size and the origin of the harvested tissue, these therapies can lead to iatrogenic complications and donor-site morbidities. In future, these side effects could be avoided by transplanting artificially generated tissue consisting of different cell types and matrix components derived from the host body. Tissue that is grown in the patient could be advantageous compared with the more simply structured in vitro-grown alternatives. To overcome the limitations of graft vascularization, the arteriovenous (AV) loop technique has been established for different tissues in the last years and was adapted for lymphatic tissue engineering in the present study. We utilized the AV loop technique to grow human lymphatic vasculature in vivo in the Rowett nude (RNU) rat. A combination of human lymphatic endothelial cells (LECs) and bone marrow-derived mesenchymal stem cells was implanted in a fibrin matrix surrounding the AV loop. After 2 or 4 weeks of implantation, the animals were perfused and the tissue was harvested. It could be demonstrated by immunohistochemistry for human LYVE1, human CD31, and murine podoplanin that the implanted cells formed human lymphatic vasculature in the AV loop chamber. Beside development of murine podoplanin-positive vasculature in the AV loop tissue, vasculature positive for human marker proteins developed in comparable numbers. This suggests that implanted LECs are able to improve the lymphatic vascularization of the newly engineered tissue. Thus, we were able to establish an in vivo tissue engineering method to generate lymphatic vascularized soft tissue. An axially vascularized transplantable lymphatic vessel network was engineered without requiring advanced cell culture equipment, rendering the lymphatic AV loop highly suitable for applied regenerative medicine. Impact statement Various surgical procedures require the transplantation of autologous harvested tissue, for example, the vascularized lymph node transfer for the treatment of lymphedema. Tissue-engineered transplants could be used instead of autologous transplants and thereby help to reduce the side effects of those therapies. However, in vitro tissue engineering of large constructs requires a lot of know-how as well as advanced cell culture equipment, which might not be accessible in every hospital. In vivo tissue engineering approaches like the presented technique for the generation of transplantable networks of lymphatic vasculature could serve as an alternative for in vitro tissue engineering approaches in clinical settings.

Alginate Hydrogels for In Vivo Bone Regeneration: The Immune Competence of the Animal Model Matters.

Biomaterials with tunable biophysical properties hold great potential for tissue engineering. The adaptive immune system plays an important role in bone regeneration. Our goal is to investigate the regeneration potential of cell-laden alginate hydrogels depending on the immune status of the animal model. Specifically, the regeneration potential of rat mesenchymal stromal cell (MSC)-laden, void-forming alginate hydrogels, with a stiffness optimized for osteogenic differentiation, is studied in 5-mm critical-sized femoral defects, in both T cell-deficient athymic Rowett Nude (RNU) rats and immunocompetent Sprague Dawley rats. Bone volume fraction, bone mineral density, and tissue mineral density are higher for athymic RNU nude rats 6 weeks postsurgery. In addition, these animals show a significantly higher number of total cells and cells with non-lymphocyte morphology at the defect site, while the number of cells with lymphocyte-like morphology is lower. Hydrogel degradation is slower and the remaining alginate fragments are surrounded by a thicker fibrous capsule. Ossification islands originating from alginate residues suggest that encapsulated MSCs differentiate into the osteogenic lineage and initiate the mineralization process. However, this effect is insufficient to fully bridge the bone defect in both animal models. Alginate hydrogels can be used to deliver MSCs and thereby recruit endogenous cells through paracrine signaling, but additional osteogenic stimuli are needed to regenerate critical-sized segmental femoral defects.

Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing.

Cell transplantation therapy utilizing neural precursor cells (NPCs) is a conceptually attractive strategy for traumatic spinal cord injury (SCI) to replace lost cells, remyelinate denuded host axons and promote tissue sparing. However, the number of mature oligodendrocytes that differentiate from typical NPCs remains limited. Herein, we describe a novel approach to bias the differentiation of directly reprogrammed human NPCs (drNPCs) toward a more oligodendrogenic fate (oNPCs) while preserving their tripotency. The oNPCs derived from different lines of human NPCs showed similar characteristics in vitro. To assess the in vivo efficacy of this approach, we used oNPCs derived from drNPCs and transplanted them into a SCI model in immunodeficient Rowett Nude (RNU) rats. The transplanted cells showed significant migration along the rostrocaudal axis and proportionally greater differentiation into oligodendrocytes. These cells promoted perilesional tissue sparing and axonal remyelination, which resulted in recovery of motor function. Moreover, after transplantation of the oNPCs into intact spinal cords of immunodeficient NOD/SCID mice, we detected no evidence of tumor formation even after 5 months of observation. Thus, biasing drNPC differentiation along an oligodendroglial lineage represents a promising approach to promote tissue sparing, axonal remyelination, and neural repair after traumatic SCI. stem cells translational medicine 2018;7:806–818

Effective Treatment of Traumatic Brain Injury in Rowett Nude Rats with Stromal Vascular Fraction Transplantation.

Traumatic brain injury (TBI) affects 1.9 million Americans, including blast TBI that is the signature injury of the Iraq and Afghanistan wars. Our project investigated whether stromal vascular fraction (SVF) can assist in post-TBI recovery. We utilized strong acoustic waves (5.0 bar) to induce TBI in the cortex of adult Rowett Nude (RNU) rats. One hour post-TBI, harvested human SVF (500,000 cells suspended in 0.5 mL lactated Ringers) was incubated with Q-Tracker cell label and administered into tail veins of RNU rats. For comparison, we utilized rats that received SVF 72 h post-TBI, and a control group that received lactated Ringers solution. Rotarod and water maze assays were used to monitor motor coordination and spatial memories. Rats treated immediately after TBI showed no signs of motor skills and memory regression. SVF treatment 72 h post-TBI enabled the rats maintain their motor skills, while controls treated with lactated Ringers were 25% worse statistically in both assays. Histological analysis showed the presence of Q-dot labeled human cells near the infarct in both SVF treatment groups; however, labeled cells were twice as numerous in the one hour group. Our study suggests that immediate treatment with SVF would serve as potential therapeutic agents in TBI.

A Xenograft Model of Vestibular Schwannoma and Hearing Loss.

Microsurgical implantation of mouse merlin-deficient Schwann cells (MD-SC) into the cerebellopontine angle of immunodeficient rats will initiate tumor formation, hearing loss, and vestibular dysfunction. The progress in identifying effective drug therapies for treatment of Neurofibromatosis type II (NF2) is limited by the availability of animal models of VS that develop hearing loss and imbalance. A microsurgical technique for implanting MD-SCs onto the cochleovestibular nerve of rats was developed. Ten Rowett Nude rats were implanted with either ∼10 MD-SCs expressing luciferase (N = 5) or vehicle (N = 5). Rats received bioluminescence imaging, auditory brainstem response testing, and were observed for head tilt every 2 weeks after surgery, for a total of 6 weeks. Tumors were harvested and processed with hematoxylin & eosin staining and immunohistochemistry was performed for S100. Rats implanted with MD-SCs developed significantly higher tumor bioluminescence measurements and hearing threshold shifts at multiple frequencies by the 4th and 6th weeks post-implantation, compared with control rats. Rats implanted with MD-SCs also developed gross tumor. The tumor volume was significantly greater than nerve volumes obtained from rats in the control group. All rats with tumors developed a head tilt, while control rats had no signs of vestibular dysfunction. Tumors demonstrated histological features of schwannoma and express S100. Using this microsurgical technique, this xenograft rat model of VS develops tumors involving the cochleovestibular nerve, shifts in hearing thresholds, and vestibular dysfunction. This animal model can be used to investigate tumor-mediated hearing loss and perform preclinical drug studies for NF2.

Development and Characterization of a Novel Congenic Rat Strain for Obesity and Cancer Research

ABSTRACTThe association between a Western Diet and colon cancer suggests that dietary factors and/or obesity may contribute to cancer progression. Our objective was to develop a new animal model of obesity and the associated pathophysiology to investigate human cancer independent of dietary components that induce obesity.A novel congenic rat strain was established by introducing the fa allele from the Zucker rat into the Rowett Nude rat to generate a “fatty nude rat”. The obese phenotype was first characterized in the new model. To then examine the utility of this model, lean and obese rats were implanted with HT-29 human colon cancer xenografts and tumor growth monitored.Fatty nude rats were visibly obese and did not develop fasting hyperglycemia. Compared to lean rats, fatty nude rats developed fasting hyperinsulinemia, glucose intolerance, and insulin resistance. Colon cancer tumor growth rate and final weight were increased (P < 0.05) in fatty nude compared to lean rats. Final tumor weight was associated with p38 kinase phosphorylation (P < 0.01) in fatty nude rats.We have established a novel model of obesity and pre-type 2 diabetes that can be used to investigate human cancer and therapeutics in the context of obesity and its associated pathophysiology.

Translating Myocardial Remuscularization.

Myocardial remuscularization can be achieved by the implantation of cardiomyocyte-containing grafts. Critical steps toward clinical translation of the first generation cardiac remuscularization therapies include the choice of animal models with high predictive validity for therapeutic outcome in clinical trials. Clinically relevant remuscularization of the failing human heart remains unachieved to date. Proof-of-concept studies in small and large animal models provide compelling evidence as to the principle feasibility of myocardial remuscularization with human cardiomyocyte grafts.1,2 Clinical trials suggest myocardial protection and preservation by cell therapeutics.3,4 The mechanisms underlying the reported therapeutic effects remain under debate, but likely include the release of protective factors (growth factors, noncoding RNA) and modulation of the disease-related inflammatory response. Further studies are needed to define the paracrine milieu hypothesis and unleash its full therapeutic potential, aiming at delaying or preventing disease progression in patients with acute and subacute myocardial injury. The implantation of cardiomyocyte grafts follows a different strategy, namely remuscularization by integration of exogenously produced cardiomyocytes. Patients with chronically scarred myocardium presenting clinically with end-stage heart failure are the primary target. The suggested plug-and-play mechanism of direct cardiac remuscularization seems straight forward, but similar as for the paracrine milieu hypothesis there is a clear need for a better understanding of how, when, and where to integrate exogenous cardiomyocytes into the failing heart to achieve optimal results. Moreover, the path toward clinical application as off-the-shelf cardiomyocyte allograft therapeutics is less defined as for point-of-care autograft formulations and small chemical or biological compounds. A challenge to direct cardiac remuscularization was for many years the limited availability of bona fide cardiomyocytes. Cardiac biopsies can be used to harvest proliferative mesenchymal cells with progenitor cell properties, but without the capacity to spontaneously convert/transdifferentiate into terminally differentiated cardiomyocytes at a therapeutically relevant scale. Induced …

Long-Term Safety of PEGylated Coagulation Factor VIII in the Immune-Deficient Rowett Nude Rat

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26- and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ~75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immune-deficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals.

Immunobiology of fibrin-based engineered heart tissue.

Different tissue-engineering approaches have been developed to induce and promote cardiac regeneration; however, the impact of the immune system and its responses to the various scaffold components of the engineered grafts remains unclear. Fibrin-based engineered heart tissue (EHT) was generated from neonatal Lewis (Lew) rat heart cells and transplanted onto the left ventricular surface of three different rat strains: syngeneic Lew, allogeneic Brown Norway, and immunodeficient Rowett Nude rats. Interferon spot frequency assay results showed similar degrees of systemic immune activation in the syngeneic and allogeneic groups, whereas no systemic immune response was detectable in the immunodeficient group (p < .001 vs. syngeneic and allogeneic). Histological analysis revealed much higher local infiltration of CD3- and CD68-positive cells in syngeneic and allogeneic rats than in immunodeficient animals. Enzyme-linked immunospot and immunofluorescence experiments revealed matrix-directed TH1-based rejection in syngeneic recipients without collateral impairment of heart cell survival. Bioluminescence imaging was used for in vivo longitudinal monitoring of transplanted luciferase-positive EHT constructs. Survival was documented in syngeneic and immunodeficient recipients for a period of up to 110 days after transplant, whereas in the allogeneic setting, graft survival was limited to only 14 ± 1 days. EHT strategies using autologous cells are promising approaches for cardiac repair applications. Although fibrin-based scaffold components elicited an immune response in our studies, syngeneic cells carried in the EHT were relatively unaffected. An initial insight into immunological consequences after transplantation of engineered heart tissue was gained through this study. Most important, this study was able to demonstrate cell survival despite rejection of matrix components. Generation of syngeneic human engineered heart tissue, possibly using human induced pluripotent stem cell technology with subsequent directed rejection of matrix components, may be a potential future approach to replace diseased myocardium.

Evaluating the Impact of Mechanical Manipulation on Inducing Disk Degeneration: A 3-Month Radiologic Follow-Up of Intervertebral Disk Degeneration in a Needle-Punctured Rat Tail Model

Introduction The rat tail model for the intervertebral disk degeneration has recently been receiving a high level of interest among the intervertebral disk degeneration studies. A better understanding of the time course and recognizing the role of mechanical manipulation may lead to development of a more reproducible model for further application in disk implanting studies. In this study, we aimed to establish a reproducible disk degeneration rat tail model by needle puncture and to assess the gradual progression of the degeneration over time. Materials and Methods Under fluoroscopic guidance, the fourth intervertebral disk space of the rat's tail in 13 mature Rowett Nude (RNU) male rats (T-Cell-Deficient, Athymic Nude Rats) was percutaneously stabbed on the left lateral side with an 18G needle. To standardize the depth of the stab, the tip of a syringe cap was cut at 5 mms point, and was then used with a new needle to puncture each new animal. The first seven rats (group 1) received no further mechanical manipulation; however in the next six rats (group 2), in addition to the needle puncture, the proximal portion of the tail including the stabbed level was then manually rotated around and compressed along to the longitudinal axis. Radiographic follow-up was carried out for 3 months; with X-rays performed at 1, 2, 4, 6, 10, and 12 weeks, and magnetic resonance imaging (MRI) scans at 4 and 12 weeks. Disk height change was evaluated by calculating the disk height index (DHI) on X-rays. In each animal, a baseline DHI was calculated based on the rostral healthy disc. Disk degeneration grade was also evaluated on T2-weighted MRI sequences based on the signal change in the annulus fibrosus and nucleus pulposus. A 1-4 modified Thompson grading system was used, defined as no, mild, moderate, and severe degeneration. Statistical analysis was performed in all animals and in the two different groups. Results On X-rays, the mean disk height in all 13 rats showed significant decrease at both 1 week and 2 weeks, with the maximum decrease observed at 2 weeks. The disk height increased gradually between 4 to 12 weeks; however it was not statistically significant. In the separate analysis of the two different groups, the second group had a similar significant decrease in disk height at 1 and 2 weeks. Despite minor changes, in the first group no statistical significance was detected. On T2-weighted MRI scans at 4 and 12 weeks, all rats had a statistically significant increase in disk degeneration grade, as compared to the baseline. However, the minor increase in degeneration grade between 4 weeks to 12 weeks was not statistically significant. On separate analysis, only the second group indicated significant increase in the disk degeneration grade after the puncture experience. The X-Ray results presented as DHIs and the MRI results described as the 1-4 modified Thompson grading system for intervertebral disk degeneration showed strong correlation in all 13 rats, and in each of the 2 groups separately. Conclusion Isolated needle puncturing of the intervertebral disk leads to significantly lower levels of degeneration compared to needle puncturing followed by further manual mechanical manipulation. The highest level of degeneration is observed at 2 weeks. Disk height decrease on X-Ray imaging and signal changes on T2-weighted MRI scans can provide good informative data on the level of degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Abstract 5310: Re-Endothelialization of Decellularized Heart Reduces Vascular Thrombogenicity

Perfusion decellularization of cadaveric hearts generates a cell-free extracellular matrix (ECM) that has similar architectural, mechanical, and chemical properties to the native tissue. These scaffolds retain their vascular conduits giving them the potential for anastomosis and transplantation. However, because the matrix is denuded, vascular clotting occurs after transplant even under an anti-coagulation (Coumadin) regimen. To create a less thrombogenic and thus more transplantable decellularized construct, we re-lined vessel conduits throughout the heart with endothelial cells and quantified their functional ability to alter clotting. Cadaveric rat hearts were decellularized by retrograde aortic detergent perfusion. Labeled rat aortic endothelial cells (RAECs) were re-introduced following two different protocols: 40 million RAECs were delivered by arterial (Ar) infusion only, or 20 million RAECs delivered by Ar infusion followed with an additional 20 million RAECs delivered by venous (Ve) infusion. Re-endotheliazed heart constructs were then maintained in a bioreactor under retrograde aortic perfusion for 1 week. Thrombogenicity of the constructs was measured by an in vitro thrombomodulin assay of the media flowing through the heart vasculature followed by histologic analysis. Heterotopic transplantation of scaffolds was performed in Rowett Nude rats, and hearts were examined 1 week post transplant. Either delivery route resulted in a whole heart (base to apex) distribution of RAECs with the retention of the endothelial phenotype (e.g. flattened morphology, proliferative (PCNA + ), eNOS + and vWF + ). In vitro thrombogenicity was reduced regardless of the delivery protocol, though there was a trend towards improved performance when cells were delivered via both Ar+Ve routes (6 and 8 fold decrease vs. controls for Ar only or Ar+Ve delivery respectively, N=8 for each condition, p&lt;0.05). After heterotopic transplantation of the re-endothelialized ECM, clotting and cellular invasion were reduced. With this advance we move closer to having a suitable heart construct for transplant while also developing a novel in vitro system for use in studies such as examining stem/progenitor cell differentiation in decellularized heart.

Human mesenchymal stem cells do not differentiate into cardiomyocytes in a cardiac ischemic xenomodel

AIM. As the capability of human mesenchymal stem cells (hMSC) to engraft, differentiate and improve myocardial function cannot be studied in humans, exploration was performed in a xenomodel.METHODS. The rats were divided into three groups depending on the type of rats used (Rowett nude (RNU) or Fischer rats +/− immunosuppression). Different groups were treated with intramyocardial injection of hMSC (1–2 million) either directly or three days after ligation of the left anterior descending artery (LAD). Myocardial function was investigated by echocardiography. The hMSC were identified with fluorescence in situ hybridization and myocardial differentiation was assessed by immunohistochemistry.RESULTS. When hMSC were injected directly after LAD ligation they could be identified in half (8/16) of the RNU rats (without immunosuppression) at 4 weeks. When injected 3 days after LAD ligation in immunosuppressed RNU rats they were identified in all (6/6) rats at 6 weeks. The surviving hMSC showed signs of differentiation into fibroblasts. No cardiomyocyte differentiation was observed. There was no difference in myocardial function in treated animals compared to controls.CONCLUSIONS. The hMSC survived in this xenomodel up to 6 weeks. However, hMSC required implantation into immunoincompetent animals as well as immunosuppression to survive, indicating that these cells are otherwise rejected. Furthermore, these cells did not differentiate into cardiomyocytes nor did they improve heart function in this xenomodel.

Biodistribution and Imaging of FDG in Rats with LS174T Carcinoma Xenografts and Focal Escherichia coli Infection

The aim of this study was to compare the dynamic distribution of fluorodeoxyglucose (FDG) in malignant and in infectious lesions. The dynamic distribution of FDG was studied in Rowett nude (RNU) rats with a LS174T carcinoma xenograft in the left front leg and an Escherichia coli-induced focal infection in the right front leg. In 5 rats, dynamic FDG-PET was performed (27 frames of 6-15 minutes) up to 4 hours after injection of 11 MBq 18FDG. The mean FDG uptake (SUV) was calculated and plotted by using a region of interest (ROI) centered over both lesions. In groups of 6 rats, the biodistribution of FDG was determined by counting dissected tissues at 1, 2, 3, and 4 hours after an injection of 11 MBq FDG. Means +/- the standard error of the mean (SEM) were calculated. Dynamic positron emission tomography (PET) visualized both the tumor and the infection. The ROI analysis showed that FDG uptake in the infections was faster and higher, as compared to the tumor lesions. FDG uptake in the tumor reached a standardized uptake value (SUV) of 0.8 +/- 0.3 at 60 minutes and in the infectious lesions a SUV of 1.6 +/- 0.2 at 45 minutes, both remaining constant until 4 hours postinjection (p.i.). In the biodistribution study with ex vivo tissue counting, FDG had accumulated up to 1.1 +/- 0.1 %ID/g and 0.8 +/- 0.1 %ID/g at 1 hour in the tumor and infection, respectively, and remained constant until 4 hours for both lesions without significantly different wash-out from the 2 lesions. The tumor/blood and abscess/ blood ratios increased with time to 57 +/- 17 and 48 +/- 14, respectively. Although in this model differences in absolute FDG uptake and initial kinetics between tumor and infection were observed, the wash-out rate of FDG from the lesions was similar over time. The retention of FDG in the inflammatory lesion indicated that dual time-point imaging does not necessarily resolve diagnostic pitfalls for FDG-PET in oncology in order to discriminate between malignant tumorous and benign infectious lesions.

Nude rats as a model of natural pneumocystis carinii pneumonia: sequential morphological study of lung lesions

A spontaneous infection with Pneumocystis carinii (P.c.) caused enzootic fatal pneumonia in a long-term experiment with athymic Rowett Nude (Han:RNU rnu/rnu) and New Zealand Nude (Han:NZNU rnuN/rnuN) rats. In order to reproduce the infection and to characterize the early pathogenesis of lung lesions, 13 young athymic Han:RNU and Han:NZNU rats from P.c.-free breeding colonies were housed together in one room with chronically P.c.-infected rats. They were killed after 8, 12, 16, 20 or 24 weeks, and their lungs were examined for P.c. infection by means of light microscopy, immunohistochemistry and transmission electron microscopy. Lung lesions progressed from a mild interstitial pneumonia with scattered alveolar macrophages (8 weeks) to a severe diffuse interstitial pneumonia with widespread areas of distended alveoli, filled with foamy material. In all lungs, P.c. antigen was detected immunohistochemically, developing from small intra-alveolar aggregations of organisms into large multifocal clusters. Ultrastructurally, P.c. trophozoites and cysts were seen attached to type I pneumocytes and lying free in the alveolar lumen. Chronic severe P.c. pneumonia (20 and 24 weeks) was characterized by masses of P.c. trophozoites in the alveoli and alveolar walls, extensive proliferation of type II pneumocytes and interstitial fibrosis. The easy and consistent horizontal transmission of spontaneous P.c. pneumonia to previously non-infected athymic Han:RNU and Han:NZNU rats and the similarity of the disease to human infection demonstrate both rat strains to be excellent models for studying pulmonary pneumocystosis of immunodeficient human patients.

Regional mapping of the Rowett nude gene (RONU) to rat chromosome 10q24→q32 by localizing linked SYB2 and GH loci

The Rowett nude gene (RONU) has been mapped on rat chromosome (Chr) 10 by linkage analysis using (ACI x F344/N-RONU/RONU)F1 x F344/N-RONU/RONU backcross progeny. The gene order on the chromosome was RR92- (16.1 cM) - RR24 - (17.9 cM) - MYHSE (myosin heavy chain, embryonic) - (1.0 cM) - SYB2 (synaptobrevin 2) - (1.0 cM) - SHBG (sex hormone-binding globulin) - (4.0 cM) - RONU (Rowett nude) - (29.0 cM) - AEP (anion exchange protein), PPY (pancreatic polypeptide) - (3.0 cM) - ACE (angiotensin I converting enzyme), GH (growth hormone). The RONU locus was localized to 10q24-->q32 by fluorescence in situ hybridization of the closely linked SYB2 and loosely linked GH loci on the opposite side. Conserved linkage of homologous loci mapped to rat Chr 10 and mouse Chr 11 supports the hypothesis that the RONU locus is a rat homolog of the mouse nu locus.

Genetic mapping of the athymic nude (RNU) locus in the rat to a region on chromosome 10.

The nude trait in the rat is transmitted in an autosomal recessive manner and is associated with thymic aplasia, T-cell deficiency, and hairlessness. Congenic rats homozygous for the RNU (Rowett nude) locus are important models in the study of inflammatory disease, tumor growth, and transplant rejection. The RNU locus has not been previously mapped, and the nature of the gene product is unknown. To determine the map location of this gene, a single F344.rnu/rnu (athymic nude congenic Fischer rat) male congenic rat was bred with 3 LEW/N (NIH stock Lewis rat) female rats to produce F1 progeny. Twelve F1 brother-sister breeding pairs were established. Forty-nine phenotypically nude F2 offspring (198 total) were obtained. Linkage analysis done on F2 DNA revealed highly significant cosegregation between the nude phenotype and eight polymorphic markers located on Chromosome (Chr) 10. The tightest linkages were with: MYH3 (embryonic, skeletal myosin heavy chain) and SHBG (sex hormone-binding globulin), giving 2 point lod scores of 20.2, and 20.0, respectively. The map order and map distances, determined by multipoint linkage calculations, were: RR24-(16.1 cM)-MYH3-(3.5 cM)-SHBG-(4.7 cM)-RNU-(11.9 cM)-F16F2-(24.1 cM)-CLATP (citrate lyase ATPase)-(2.4 cM)-ACE (angiotensin converting enzyme)/PPY (pancreatic polypeptide)-(14.1 cM)-RR1023. The position of the RNU locus in the rat corresponds closely with that of the recently reported nu locus in the mouse. This finding suggests that the nude phenotype in the rat and the mouse arise from defects in homologous genes.

The Rowett nude (rnu) rat as a host in the popliteal lymph node GVH reaction.

When parental lymphocytes are injected into the footpad of an F1 hybrid recipient, the draining popliteal lymph node increases in size and cellularity over the next 7 days. This regional GVH reaction is initiated by donor T cells (specifically the W 3/25 positive subset) recognizing the foreign transplantation antigens of the host (Ford et al., 1970; Brideau et al., 1980). Nevertheless, the increased cellularity of the lymph node is mainly due to the accumulation and division of T and B lymphocytes of host origin (Rolstad and Fossum, 1977). The nature of the link between antigen recognition by donor cells and the activation of host cells into proliferation is not known. Apparently it does not require the presence of host T cells, since the popliteal lymph node response was of similar magnitude in normal and T cell depleted animals (Rolstad, 1976).