Evaluation of Simulated Weightlessness Model of Hindlimb Unloading Miniature Pigs and Their Tissue Damage

doi:10.12300/j.issn.1674-5817.2024.038

Abstract

Abstract:

Objective To establish a weightlessness simulation animal model using miniature pigs, leveraging the characteristic of multiple systems’ tissue structures and functions similar to those of humans, and to observe pathophysiological changes, providing a new method for aerospace research. Methods Nine standard-grade miniature pigs were selected and randomly divided into an experimental group (n=7) and a control group (n=2). The experimental group was fixed using customized metal cages, with canvas slings suspending their hind limbs off the ground, and the body positioned at a -20° angle relative to the ground to simulate unloading for 30 days (24 hours a day). Data on body weight, blood volume, and blood biochemistry indicators were collected at different time points for statistical analysis of basic physiological changes. After the experiment, the miniature pigs were euthanized and tissue samples were collected for histopathological observation of the cardiovascular, skeletal and muscle systems HE and Masson staining. Statistical analysis was also conducted on the thickness of arterial vessels and the diameter of skeletal muscle fibers. Additionally, western blotting was employed to detect the expression levels of skeletal muscle atrophy-related proteins, including muscle-specific RING finger protein 1 (MuRf-1) and muscle atrophy F-box (MAFbx, as known as Atrogin-1), while immunohistochemistry was used to detect the expression of glial fibrillary acidic protein (GFAP), an indicator of astrocyte activation in the brain, reflecting the pathophysiological functional changes across systems. Results After hindlimb unloading, the experimental group showed significant decreases in body weight (P<0.001) and blood volume (P<0.01). During the experiment, hemoglobin, hematocrit, and red blood cell count levels significantly decreased (P<0.05) but gradually recovered. The expression levels of alanine aminotransferase and γ-glutamyltransferase initially decreased (P<0.05) before rebounding, while albumin significantly decreased (P<0.001) and globulin significantly increased (P<0.01). Creatinine significantly decreased (P<0.05). The average diameter of gastrocnemius muscle fibers in the experimental group significantly shortened (P<0.05), with a leftward shift in the distribution of muscle fiber diameters and an increase in small-diameter muscle fibers. Simultaneously, Atrogin-1 expression in the gastrocnemius and paravertebral muscles significantly increased (P<0.05). These changes are generally consistent with the effects of weightlessness on humans and animals in space. Furthermore, degenerative changes were observed in some neurons of the cortical parietal lobe, frontal lobe, and hippocampal regions of the experimental group, with a slight reduction in the number of Purkinje cells in the cerebellar region, and a significant enhancement of GFAP-positive signals in the hippocampal area (P<0.05). Conclusion Miniature pigs subjected to a -20° angle hind limb unloading for 30 days maybe serve as a new animal model for simulating weightlessness, applicable to related aerospace research.

Key words: Simulated weightlessness, Miniature pig, Animal model, Hindlimb unloading

CLC Number:

R-332

TU Yingxin,JI Yilan,WANG Fei,et al. Evaluation of Simulated Weightlessness Model of Hindlimb Unloading Miniature Pigs and Their Tissue Damage[J]. Laboratory Animal and Comparative Medicine, 2024, 44(5): 475-486. DOI: 10.12300/j.issn.1674-5817.2024.038.

Figures/Tables 6

Figure 1 Experimental group miniature pigs fixed in a customized metal cage for hindlimb unloading simulated weightlessness experiment

Figure 2 Changes in selected basic physical indicators of miniature pigs in the experimental and control groups during hindlimb unloading simulated weightlessness experimentNote：Miniature pigs in the experimental group (n=7) were immobilized using customized metal cages and suspended from canvas slings so that their hind limbs were off the ground to unload and their bodies were at a -20° angle to the ground. Control group miniature pigs (n=2) were housed in metal cages without suspension immobilization. A, Changes in body weight during the experiment (*** the body weight change of the experimental group is significantly different from the control group）; B, Changes in red blood cell count (RBC) during the experiment; C, Changes in hemoglobin（HGB） during the experiment; D, Changes in hematocrit (HCT) during the experiment; E, Changes in plasma volume change (△PV) during the experiment (** the ΔPV of experimental group is significantly higher than the control group at the 8th day); F, Changes in serum alanine aminotransferase (ALT) during the experiment; G, Changes in serum γ-glutamyl transferase (γ-GT) during the experiment; H, Changes in serum albumin (ALB) during the experiment; I, Changes in serum globulin (GLB) during the experiment; J, Changes in creatinine (Cr) during the experiment. *P<0.05， **P<0.01， ***P<0.001.

Figure 3 Histopathology and thickness changes of arterial blood vessels in miniature pigs after the hindlimb unloading simulated weightlessness experimentNote：Miniature pigs in the experimental group (n=7) were immobilized using customized metal cages and suspended from canvas slings so that their hind limbs were off the ground to unload and their bodies were at a -20° angle to the ground. Control group miniature pigs (n=2) were housed in metal cages without suspension immobilization. A, HE staining of each artery (the scale size is 100 μm); B, Masson staining of each artery (the scale size is 100 μm); C, Statistical analysis of intima-media thickness in each artery.

Figure 4 Changes in skeletal muscle structure and muscle fiber diameter of miniature pigs after the hindlimb unloading simulated weightlessness experimentNote：Miniature pigs in the experimental group (n=7) were immobilized using customized metal cages and suspended from canvas slings so that their hind limbs were off the ground to unload and their bodies were at a -20° angle to the ground. Control group miniature pigs (n=2) were housed in metal cages without suspension immobilization. Figures A, B, and C represent the HE staining images (the scale size is 100 μm or 20 μm by low and high magnification) , the statistical chart of muscle fiber diameters, and the distribution chart of muscle fiber diameters for various skeletal muscle tissues (gastrocnemius muscle, soleus muscle, paravertebral muscle, and forelimb muscle),respectively. *P<0.05.

Figure 5 Changes in expression levels of muscle atrophy proteins MuRf-1 and Atrogin-1 in miniature pigs after the hindlimb unloading simulated weightlessness experimentNote：Miniature pigs in the experimental group (n=7) were immobilized using customized metal cages and suspended from canvas slings so that their hind limbs were off the ground to unload and their bodies were at a -20° angle to the ground. Control group miniature pigs (n=2) were housed in metal cages without suspension immobilization. A, Expression levels of MuRf-1 and Atrogin-1 in soleus muscle and gastrocnemius muscle detected using western blotting; B, Expression levels of MuRf-1 and Atrogin-1 in paravertebral muscle and forelimb muscle detected using western blotting. *P<0.05， **P<0.01.

Figure 6 Changes in histopathology and related protein expression in each brain region of miniature pigs after the hindlimb unloading simulated weightlessness experimentNote：Miniature pigs in the experimental group (n=7) were immobilized using customized metal cages and suspended from canvas slings so that their hind limbs were off the ground to unload and their bodies were at a -20° angle to the ground. Control group miniature pigs (n=2) were housed in metal cages without suspension immobilization. A, HE staining of each brain region (the scale size is 20 μm), the arrow indicates the degenerating neuron; B, Glial fibrillary acidic protein (GFAP) immunohistochemistry staining of each brain region (the scale size is 50 μm); C, Quantitative analysis of GFAP expression in each brain region (IOD, integrated optical density).**P<0.01.

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