Tibet Minipigs Research Articles

TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway

BackgroundMesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation.MethodPrimary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs’ proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-β-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs.ResultsTZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis.ConclusionsThis study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.

Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic Microinjection.

The Tibet minipig is a rare highland pig breed worldwide and has many applications in biomedical and agricultural research. However, Tibet minipigs are not like domesticated pigs in that their ovulation number is low, which is unfavourable for the collection of zygotes. Partly for this reason, few studies have reported the successful generation of genetically modified Tibet minipigs by zygote injection. To address this issue, we described an efficient way to generate gene-edited Tibet minipigs, the major elements of which include the utilization of synchronized oestrus instead of superovulation to obtain zygotes, optimization of the preparation strategy, and co-injection of clustered regularly interspaced short palindromic repeat sequences associated protein 9 (Cas9) mRNA and single-guide RNAs (sgRNAs) into the cytoplasm of zygotes. We successfully obtained allelic TYR gene knockout (TYR-/-) Tibet minipigs with a typical albino phenotype (i.e., red-coloured eyes with light pink-tinted irises and no pigmentation in the skin and hair) as well as TYR-/-IL2RG-/- and TYR-/-RAG1-/- Tibet minipigs with typical phenotypes of albinism and immunodeficiency, which was characterized by thymic atrophy and abnormal immunocyte proportions. The overall gene editing efficiency was 75% for the TYR single gene knockout, while for TYR-IL2RG and TYR-RAG1 dual gene editing, the values were 25% and 75%, respectively. No detectable off-target mutations were observed. By intercrossing F0 generation minipigs, targeted genetic mutations can also be transmitted to gene-edited minipigs' offspring through germ line transmission. This study is a valuable exploration for the efficient generation of gene-edited Tibet minipigs with medical research value in the future.

The effects of irreversible electroporation on the stomach wall after ablating hepatic tissues.

This study aimed to evaluate the effect of irreversible electroporation (IRE) on the stomach wall after IRE was applied on liver tissues adjacent to the anterior wall of the stomach. IRE ablation was performed in eight Tibet mini-pigs with three lesions per pig. The IRE electrodes were inserted into the liver tissues situated close to the anterior wall of the stomach. As for the control group, the IRE electrodes were also inserted into the liver tissues for three lesions in four Tibet mini-pigs but did not turn on the current. Serum aminotransferase and WBC levels clearly increased in all the IRE ablated animals by Day 1 and decreased gradually thereafter. The gross postmortem examination at 7days post-IRE revealed a whitish lesion with sharp demarcation on the serosal surface of the stomach, but we could not find any signs of ablation or just find a small, slightly reddish lesion at the Day-28 examination. On the Day-7 histopathological examination, inflammation and fibrosis were observed in the serosal layer of the stomach in each animal and mild inflammation of the myofibers was found in only two pigs. All the stomach layers returned to normalcy by 28days post-IRE. Thus, IRE ablation of hepatic tissues situated close to the stomach wall cannot lead to stomach perforation. IRE is therefore a safe procedure for ablating hepatic tumors that are adjacent to the stomach.

The diagnostic value of [18F]-FDG-PET/CT in assessment of radiation renal injury in Tibet minipigs model

BackgroundRadiation-induced kidney damage can severely affect renal function, and have a serious impact on glucose reabsorption. Fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is routinely utilized for metabolic imaging of glucose utilization. In this study, we are trying to assess the diagnostic value of 18F-FDG-PET/CT on measuring hyperacute effect of total body irradiation (TBI) on the kidneys.MethodsForty-eight Tibet minipigs were treated by TBI of different dosages using an 8-MV X-ray linear accelerator. Whole-body 18F-FDG-PET/CT was performed at 6, 24 and 72 h followed by histologic examination, blood samples’ and renal function analysis.ResultsThe uptake of 18F-FDG was significantly different between 11/14 Gy dose groups and control group, the standard Uptake Values reached a maximal level at 72 h after 14-Gy TBI treatment. At doses over 8 Gy, histological observation showed formation of tube casts, degeneration, necrosis of tubular cells, inflammatory cell infiltration and dilatation of the mitochondria of tubule cells. Renal function analysis confirmed the changes in blood urea nitrogen and creatinine levels at various dosages and time intervals. Immunohistochemistry and western blot results indicate that the expression levels of IL-10 and TNF-α proteins were positively correlated with radiation dose up to 8 Gy.Conclusions18F-FDG PET/CT can reflect pathological changes in kidneys and it may be a useful tool for rapid and non-invasive assessment in cases of suspected radiation-induced kidney damage.

Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer (SCNT).

Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.

Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet-rich plasma.

Macroporous calcium phosphate cement (CPC) with stem cell seeding is promising for bone regeneration. The objective of this study was to investigate the effects of co-delivering autologous bone marrow mesenchymal stem cells (BMSCs) and autologous platelet-rich plasma (PRP) in CPC scaffold for bone regeneration in minipigs for the first time. Twelve female adult Tibet minipigs (12-18months old) were used. A cylindrical defect with 10mm height and 8mm diameter was prepared at the femoral condyle. Two bone defects were created in each minipig, one at each side of the femoral condyle. Three constructs were tested: (1) CPC scaffold (CPC control); (2) CPC seeded with BMSCs (CPC-BMSC); (3) CPC seeded with BMSCs and PRP (CPC-BMSC-PRP). Two time points were tested: 6 and 12weeks (n=4). Good integration of implant with surrounding tissues was observed in all groups. At 12weeks, the CPC-BMSC-PRP group had significantly less residual CPC remaining in the defect than the CPC-BMSC group and the CPC control (p<0.05). The residual CPC volume for the CPC-BMSC-PRP group was half that of the CPC control. New bone formation for CPC-BMSC-PRP was more than two-fold that of the CPC control (p<0.05). CPC-BMSC-PRP had new blood vessel density that was nearly two-fold that of the CPC control (p<0.05). In conclusion, CPC scaffold with autologous BMSC-PRP doubled the new bone regeneration and blood vessel density in minipigs compared with the CPC control. In the present study, the new macroporous CPC system with co-delivered BMSC-PRP has been shown to promote scaffold resorption and bone regeneration in large defects. Copyright © 2017 John Wiley & Sons, Ltd.

The Effects of Irreversible Electroporation on the Colon in a Porcine Model.

Background and AimIrreversible electroporation (IRE) is a method of targeted cell ablation which has been suggested as a potential cancer therapy as it leaves structures such as blood vessels and the extracellular matrix intact, thereby allowing the rapid recovery of healthy tissue. Here, we investigated the effects of IRE on the colon in vivo in a porcine model.MethodsIRE ablation was performed on the colon walls of 12 female Tibet mini-pigs, creating a total of 24 lesions. Lesions were monitored periodically by endoscopy. The pigs were euthanized 7, 14, 21 or 28 days after IRE ablation and the colons harvested for gross and histological analysis. Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome (MT) stain and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.ResultsAll pigs tolerated the ablation procedure without serious clinical symptoms or complications. There was no evidence of perforation by endoscopy or gross postmortem examination. All lesions were characterized by necrotic cell death with mild inflammation and hyperemia, with a sharp demarcation between ablated and adjacent normal tissue. A fibrous scar was observed in the ablated colon tissue. Histological analysis revealed damage to each layer of the colon. Histopathology findings also showed the preservation of extracellular structures and the recovery of the ablated colon.ConclusionsThe complete ablation of the target area, its rapid recovery and the lack of posttreatment symptoms suggest that IRE ablation may be a promising therapy for tumors located adjacent to or violating the colon wall.

Diagnostic value of [ $$^{18}{\text{F}}$$ 18 F ]FDG-PET/CT in acute hepatic radiation toxicity: a Tibet minipig model

There are few reports with respect to the details of acute effects (during and following RT) of ionizing radiation in liver tissue in epidemiology, pathology, physiology and imageology. Therefore, this study was undertaken to assess the diagnostic value of 2-[\({}^{18}\hbox {F}\)]-fluoro-2-deoxy-D-glucose positron emission tomography with computed tomography ([\({}^{18}\hbox {F}\)]-FDG-PET/CT) in the detection of acute radiation toxicity in normal liver using Tibet minipigs as a model. Thirty-six male Tibetan minipigs were randomly divided into six groups (\(n = 6\)). The irradiation groups were treated with a single dose of \(2, 5, 8, 11 \hbox { and } 14 \hbox { Gy}\) total body irradiation using an 8-MV X-ray linac, at dose rate of \(255 \hbox { cGy/min}\). The control group was not irradiated. The pigs were evaluated with [\({}^{18}\hbox {F}\)]-FDG-PET/CT, and their alanine aminotransferase (AST) and aspartate aminotransferase (ALT) activities and the ALT/AST ratio, INRatio and bilirubin were measured on Day 7 post-TBI. All pigs were killed on Day 7 post-TBI to collect liver tissues for pathological examination. The results showed that liver standardized uptake value (SUV) increased with the dose from 2 to 11 Gy and decreased suddenly at \(14 \hbox { Gy}\). HAI score showed a radiation dose-dependent increase of 2–11 Gy and was positively correlated with SUV (\(p<0.05\)). However, HAI score showed no correlation with liver function. Therefore, we concluded that [\({}^{18}\hbox {F}\)]-FDG-PET/CT has the potential to assess acute radiation-induced hepatic injury on early stage.

Establishment of the tibet mini-porcine model of coronary stent implantation and long term observation

Objective To establish Tibet mini-pig' s coronary stent implantation and reexamination model for interventional device valuation.Methods By using femoral artery puncture method,50 stents were planted into coronary arteries of 25 Tibet mini-pigs.Once the stent was deployed,quantitative coronary angiography and intravascular ultrasound examination were performed immediately and repeated on the end of the 1 st month and 6th month after stent implantation.Part of these pigs were sacrificed to obtain the arteries with stents for histologic analysis.Results Due to ventricular fibrillation,a pig died before stent implantation.The implantations of the rest mini-pigs were successful,with success rate of implantation up to 96％.The adoption of ketamine,midazolam and scopolamine for induced anesthesia had faster action and positive effect,about (4.5 ± 1.5) min; and the adoption of combined intravenous anesthesia of ketamine and propofol during operation had positive anesthetic effect with less adverse reactions.The ligated femoral artery could be separated again during reexamination so as to conduct angiography and ultrasonic examination.Conclusion The use of Tibet mini-pig as an animal model for evaluation of percutaneous coronary intervention apparatus is easy to be implemented and replicated. Key words: Tibet mini-pigs; Coronary; Stent ; Angiography ; Model, animal

Effects of simvastatin on heat hock protein 70 after spinal cord ischemia reperfusion injury

Objective To investigate the protective effect of simvastatin on spinal cord ischemia/reperfusion injury through the observation of the changes of heat shock protein 70 (Hsp70).Methods 24healthy adult Tibet mini-pigs of either sex,weighted 20-30 kg,were randomly divided into three groups (n =8),including sham operation group (group C),ischemia/reperfusion group (group I/R) and simvastatin experimental group (group Sim).In group I/R and Sim,the models of spinal cord ischemia/reperfusion were established by the occlusion of thoracic aorta and subclavian artery (70 min).The accessory hemiazygos vein was isolated and the distal end was ligated.In group I/R,1000 ml lactated ringer solution were pumped into the vein and the rate was 860 ml/h during the ischemia time.In group Sim,1000 ml simvastatin solution were pumped into the vein and the dose was 0.25 mg/kg during the ischemia time.Feed the swines of the experimental group simvastatin tablets 80 mg/d,orally for 30 days.After the operation,the motor function scores were recorded at 6,24,48 h (see Taylor motor function scoring system).All eight swines of each group were killed at 30 days after reperfusion,taking the L2-L5 and sacral spinal cord as quickly as possible.The hematoxylin and eosin (HE) staining method and the NISSLE staining method were adopted to observe the morphological changes of neurons and the immunohistochemical method was adopted to detect the changes of Hsp70.Results In group Sim,the 24,48 h motor function scores (3.2 ± 0.3,3.6 ± 0.2 respectively) after operation were significantly higher than that in group I/R (2.2 ±0.4,2.8 ± 0.4 respectively) (P ＜ 0.05).Compared with group I/R,the morphology of neurons in group Sim had a slight damage and higher expression changes of Hsp70 can be found in group Sim (P ＜ 0.05).Conclusion Simvastatin has a significant protective effect on the spinal cord ischemia reperfusion injury and the potential mechanism may be related to the up-regulation of Hsp70. Key words: Simvastatin; Spinal cord; Ischemia/reperfusion injury; Heat shock protein 70; Protection

A novel micro-controlled dynamic intramedullary nail for tibial middle fracture in Tibet mini-pigs

Objective To explore the effect on the fracture union in Tibet mini-pigs of a novel micro-controlled dynamic intramedullary nail which was self-designed on the basis of anatomic parameters of the animal tibia.Methods We measured the length,arc and inner diameters of the starting,narrowest and ending points of the bilateral tibial medullary cavities on the X-ray films of 20 Tibet mini-pigs.We designed and manufactured a new micro-controlled intramedullary nail using UG software on the basis of the anatomic measurements and CT 3D reconstructions of the animal tibia.The new fixation nails were tested in the animal models of left tibial middle fracture in 10 Tibet mini-pigs which were randomized into 2 groups (n =5).The experiment group was treated with the novel micro-controlled dynamic intramedullary nail and the control group with the static interlocking intramedullary nail.The fracture union was evaluated and compared between the 2 groups by X-ray,gait analysis and micro CT.Results Of the medullary cavity,the triangle proximal end was expansive,the middle segment was narrow and the oval distal end was also expansive in the Tibet mini-pigs.The length of medullary cavity averaged 113.31 ± 9.56 mm and the cavity arc 25.66° ± 3.37°.The nail length was 90 to 130 mm,the nail diameter 6 to 9 mm,the arc 15° to 25°,and the diameter of both locked screw and bioresorbable material 4.0 mm.The micro CT showed that the experimental group had significantly higher volume bone density (555.47 ± 23.49 mg/mm3) and bone volume fraction (0.84 + 0.04) than the control group (494.45 ± 16.62 mg/mm3 and 0.78 ± 0.03 respectively) (P ＜ 0.05).Conclusion It may help fracture union to design a novel micro-controlled dynamic intramedullary nail based on the theory of micro-controlled motion in clinic research. Key words: Bone nails; Fracture fixation, intramedully; Fracture healing; Imaging,three-dimensional

Research Note Polymorphism analysis of IGFBP-5 gene exon 1 in Tibet Mini-pig and Junmu No. 1 White pig

The genetic resources and the mechanism of miniaturization in the Tibet Mini-pig have not been comprehensively studied. Polymorphisms in genes related to the insulin-like growth factor (IGF) axis have been investigated for years, but few on the polymorphism of IGF-binding protein-5 (IGFBP-5) in the Tibetan pig. In this study, allele-specific polymerase chain reaction (AS-PCR) was used to analyze polymorphisms in exon 1 of the IGFBP-5 gene in two pig breeds, Tibet Mini-pigs and Junmu No. 1 White pigs. A BLAST analysis of the expressed sequence tags in the porcine IGFBP-5 gene revealed that exon 1 of this gene has two single nucleotide polymorphisms (SNPs), G188T and G503A. The AS-PCR results demonstrated that in both pig breeds examined, the TT, GT, and GG genotypes existed at the G188T locus, with GT as the most common genotype. At the G503A locus, GG, GA, and AA genotypes existed in Junmu No. 1 White pigs, with the GA genotype as the most frequently occurring. By contrast, at this locus, only the GA and AA genotypes were observed in the Tibetan pigs, and AA was more common than GA. There was a significant difference (P < 0.01) in allele distribution between the two breeds at the G503A locus but not the G188T locus, and there was a lower polymorphism information content for the two polymorphic loci in Tibet Mini-pigs than in Junmu No. 1 White pigs. The present study revealed SNPs in exon 1 of IGFBP-5 gene in the Tibet Mini-pig, possibly providing more understanding of the mechanism of miniaturization.

Single Intraperitoneal Injection of Monocrotaline as a Novel Large Animal Model of Chronic Pulmonary Hypertension in Tibet Minipigs

ObjectiveThe purpose of this study was to establish an animal model of chronic pulmonary hypertension with a single-dose intraperitoneal injection of monocrotaline (MCT) in young Tibet minipigs, so as to enable both invasive and noninvasive measurements and hence facilitate future studies.MethodsTwenty-four minipigs (8-week-old) were randomized to receive single-dose injection of 12.0 mg/kg MCT (MCT group, n = 12) or placebo (control group, n = 12 each). On day 42, all animals were evaluated for pulmonary hypertension with conventional transthoracic echocardiography, right heart catheterization (RHC), and pathological changes. Findings of these studies were compared between the two groups.ResultsAt echocardiography, the MCT group showed significantly higher pulmonary arterial mean pressure (PAMP) compared with the controls (P<0.001). The pulmonary valve curve showed v-shaped signals with reduction of a-waves in minipigs treated with MCT. In addition, the MCT group had longer pulmonary artery pre-ejection phases, and shorter acceleration time and ejection time. RHC revealed higher mean pulmonary arterial pressure (mPAP) in the MCT group than in the control group (P<0.01). A significant and positive correlation between the mPAP values and the PAMP values (R = 0.974, P<0.0001), and a negative correlation between the mPAP and ejection time (R = 0.680, P<0.0001) was noted. Pathology demonstrated evidence of pulmonary vascular remodeling and higer index of right ventricular hypertrophy in MCT-treated minipigs.ConclusionA chronic pulmonary hypertension model can be successfully established in young minipigs at six weeks after MCT injection. These minipig models exhibited features of pulmonary arterial hypertension that can be evaluated by both invasive (RHC) and noninvasive (echocardiography) measurements, and may be used as an easy and stable tool for future studies on pulmonary hypertension.

Irradiation Induced Injury Reduces Energy Metabolism in Small Intestine of Tibet Minipigs

BackgroundThe radiation-induced energy metabolism dysfunction related to injury and radiation doses is largely elusive. The purpose of this study is to investigate the early response of energy metabolism in small intestinal tissue and its correlation with pathologic lesion after total body X-ray irradiation (TBI) in Tibet minipigs.Methods and Results30 Tibet minipigs were assigned into 6 groups including 5 experimental groups and one control group with 6 animals each group. The minipigs in these experimental groups were subjected to a TBI of 2, 5, 8, 11, and 14 Gy, respectively. Small intestine tissues were collected at 24 h following X-ray exposure and analyzed by histology and high performance liquid chromatography (HPLC). DNA contents in this tissue were also examined. Irradiation causes pathologic lesions and mitochondrial abnormalities. The Deoxyribonucleic acid (DNA) content-corrected and uncorrected adenosine-triphosphate (ATP) and total adenine nucleotides (TAN) were significantly reduced in a dose-dependent manner by 2–8 Gy exposure, and no further reduction was observed over 8 Gy.ConclusionTBI induced injury is highly dependent on the irradiation dosage in small intestine and inversely correlates with the energy metabolism, with its reduction potentially indicating the severity of injury.

The diagnostic value of [18F]-FDG-PET/CT in hematopoietic radiation toxicity: a Tibet minipig model

This study was undertaken to assess the diagnostic value of 2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography with computed tomography ([18F]-FDG-PET/CT) in the detection of radiation toxicity in normal bone marrow using Tibet minipigs as a model. Eighteen Tibet minipigs were caged in aseptic rooms and randomly divided into six groups. Five groups (n = 3/group) were irradiated with single doses of 2, 5, 8, 11 and 14 Gy of total body irradiation (TBI) using an 8-MV X-ray linear accelerator. These pigs were evaluated with [18F]-FDG-PET/CT, and their marrow nucleated cells were counted. The data were initially collected at 6, 24 and 72 h after treatment and were then collected on Days 5–60 post-TBI at 5-day intervals. At 24 and 72 h post-TBI, marrow standardized uptake value (SUV) data showed a dose-dependent decrease in the radiation dose range from 2–8 Gy. Upon long-term observation, SUV and marrow nucleated cell number in the 11-Gy and 14-Gy groups showed a continuous and marked reduction throughout the entire time course, while Kaplan–Meier curves of survival showed low survival. In contrast, the SUVs in the 2-, 5- and 8-Gy groups showed early transient increases followed by a decline from approximately 72 h through Days 5–15 and then normalized or maintained low levels through the endpoint; marrow nucleated cell number and survival curves showed approximately the same trend and higher survival, respectively. Our findings suggest that [18F]-FDG-PET/CT may be helpful in quickly assessing the absorbed doses and predicting the prognosis in patients.

Effect of Total Body X-ray Irradiation on Lymph Node in Tibet Minipig

The purpose of this study was to determine the time-dose-effect of total body X-ray irradiation on lymphocytes in lymph nodes and peripheral blood in Tibet minipigs. Forty-eight Tibet minipigs were assigned into 6 groups including 5 experimental groups with 9 and the control group with 3. The minipigs in experimental groups were subjected to a total body X-ray irradiation of 2, 5, 8, 11, and 14 Gy respectively. Lymph nodes and peripheral blood samples were collected at 6, 24, and 72 hours after X-ray exposure and received histological microscopy examination and apoptosis analysis. Histology observation showed that the number of lymphocytes decreased within the lymph nodes with the increase of radiation doses and exposure time. The observation of transmission electron microscopy (TEM) showed typical apoptotic cells below 11 Gy while at 14 Gy necrotic cells were dominant. The apoptotic rate of lymphocytes in the lymph nodes was positively correlated with radiation dose in the range of 2-11 Gy, and reached the maximal level (39.4 ± 2.8) at 24 hours after 11 Gy irradiation, followed by a decrease in the apoptotic rate, but still higher than that of the control group. The number of lymphocytes in the peripheral blood samples was decreased significantly by increasing of the radiation dose and exposure time. We conclude that early damage of lymphocytes by total body X-ray irradiation is dose and time dependent below 11 Gy and before 24 hours post irradiation, and that the dosage of irradiation less than 11 Gy induced apoptosis, whereas the dose at 14 Gy resulted in necrosis in lymphocytes of the lymph nodes.

Empirical study of autogenous dermal grafts in the prevention of epidoral fiSrosis and adhesions

Objective To investigate the ability of auto-dermis grafts in the prevention of epidural fibrosis and adhesions, and provide reference for clinical application. Methods Five Tibet mini-pigs were used and laminectomy was performed in lumbar 2 and 4 segment. Dura was exposed and covered with auto-genous dermis grafts in lumbar 2 (the experimental group), without grafts in lumbar 4 (the autogeoous blank group). The specimens were observed grossly and histologically at 2, 4, 6, 8 and 10 weeks postoperatively. Epidural scar amount and adhere range was evaluated by modified Robertson scoring method and analyzed by SPSS 13.0. No sear and adhesion, 0 point; small amount scar and light-range adhesion, 1 point; medium sear and midrange adhesion, 2 pionts; bulk scar ang high-range adhesion, 3 pionts. Results All grafted dermis survived and thicken obviously than dermis in body surface (P< 0.05). Hair growth, sebaceous gland cyst and sweat cystogenesis were not observed. A larvaceous dissection abruption plane, little cicatricial tis-sue and adhesions rarefaction between dermis and dura were observed in the experimental group. Massive ei-catricial tissue from erector spinae filled in cavitas epiduralis was observed and dura was separated difficult-ly from cicatricial tissue in the autogeous blank group. Epidural scar amount and adhesion range in the ex-perimental group was significantly lower than the blank group (P< 0.05). Conclusion Dermis is a kind of favourable biomaterial with restraining cicatrisation and isolation barrier. Autogenous dermis grafts has the best effect on preventing peridural cicatrisation and adhesion after laminectomy. Key words: Dermis; Transplantation, autologons; Dura mater