Establishment Of The Second Degree Scald Model And Study On Inflammatory Response Of Germ-free Rats

Objective:Germ-free rats were obtained by Caesarean section of SPF grade Wistar rats and artificial sterile feeding technique.We established a deep second-degree scald injury model and model with Pseudomonas aeruginosa infection with these germ-free rats,and we also established a deep second-degree scald injury model with SPF rats.Then we compared the similarities and differences between the three groups in terms of healing process,inflammatory response,and pathological changes,and explored the effect of inflammatory reaction in the healing process of burn wounds.Contents:The rat isolator was placed in a barrier environment and was sterilized with 15%peracetic acid at 7 mL/m³ plus 7 mL of water for 2 hours to obtain a sterile environment.The SPF Wistar rats were caesareanized in a sterile environment to obtain sterile neonate rats,and artificially sterilized with artificial milk until the neonate rats could eat independently.The first generation of offspring of these sterile neonate rats was raised by breastfeeding.A rat model with deep second-degree scald was established in a clean bench by a constant temperature and pressure scald apparatus in the6-week old germ-free rat of the offspring.The burned germ-free rats were randomly divided into two groups.One group was infected with pseudomonas aeruginosa at a concentration of 1×10⁵ CFU/mL at the wound site,and one group was not treated.At the same time,SPF-level scald rats were set up as controls.The healing process,inflammatory reaction and pathological changes of the three groups were observed to analyze the effects of Pseudomonas aeruginosa and inflammatory reaction on wound healing after burn.Methods:Ten week old male and female SPF wistar rats were caged and mated in a ratio of 2:1.After 21 days pregnant,female rats were given caesarean sections in a rat isolator.These aseptic neonate rats were given artificial milk 6 times daily from 1 to 14 days and then 4 times daily from 15 to21 days after birth until they could eat independently.During the growth process,any items introduced through the transfer window into the isolator are sterilized to ensure a sterile environment.Feed,drinking water,surgical instruments,cages and beakers were autoclaved at 121°C for 30 minutes.Goat milk,a portion of Abbott’s infant formula,and newborn calf serum were sterilized with ⁶⁰Co.The brain heart broth,sodium thioglycolate,and blood agar plate were used to incubate sterile physiological saline washing solution of embryos and uterus that obtained from pregnant rats in a constant temperature incubator at 37°C for 14 days.No bacteria were detected.The soybean peptone broth was cultured in a constant temperature incubator at26°C for 7 days and eumycete wasn’t detected.The feces dilution,throat swab of the neonate rats and isolator were detected every two weeks and no bacterium was detected.These rats were copulated when they were 10 weeks old.The first generations of offspring of these rats were tested as no bacteria affected.The germ-free rat was obtained.Deep second degree scald wound was performed in a six weeks old female germ-free rat weighted 160¹80g at a temperature of 94°C,8s and pressure 500g in a clean bench.The scald germ-free rats were randomly divided into two groups.One group was infected with pseudomonas aeruginosa in the wound;another group was not infected and the wound kept clean.SPF-level scald wistar rats were set up as control group under the same conditions.The scab generating time,dislocation time and healing time of the three groups were observed,and serum samples were taken to detect the changes of inflammatory cytokines such as GM-CSF,IL-1αand TNF-α.The skin,lung,pancreas,liver and kidney tissues were fixed in 4%formaldehyde solution and used for histopathology at 24 hours,3 days and 7days after scald.Seven days after scald,the plate count method was used to measure the number of wound flora.Results:1 Preparation of germ-free ratsGerm-free rats were successfully obtained and have fertility transfer ability2 The establishment of deep second degree scald modelInnovative scalding method can get deep second degree scald model.3 The healing process of three groups of ratsCompared with aseptic scald rats,the pseudomonas aeruginosa stained rats had a longer period of scab generating time with significant differences（P<0.05）;the decrustation and healing time was shorter than that of germ-free rats.And there was a significant difference（P<0.05）.4 The inflammatory reaction of the three groups4.1 Changes of TNF-αlevels in serumThe serum level of TNF-αin three groups of scalded rats was higher than that before burned,and there was a significant difference（P<0.05）.There was significant difference in serum TNF-αbetween the three groups at 24h （P<0.05）,the serum TNF-αof scald rats with Pseudomonas aeruginosa was significantly lower than the SPF group and the aseptic group.4.2 Changes of GM-CSF levels in serumThe serum level of GM-CSF in three groups of scalded rats was higher than that before burned,and there was a significant difference（P<0.05）.Serum GM-CSF was significantly different between the three groups at 24 hours（P<0.05）,the scald burn aseptic group was significantly higher than the scald burn group with Pseudomonas aeruginosa and SPF group.At the third days,Serum GM-CSF was significantly different（P<0.05）,the scald burn aseptic group was significantly higher than the scald burn group with Pseudomonas aeruginosa.At the seventh days,Serum GM-CSF was significantly different（P<0.05）,the scald burn aseptic group was significantly lower than the scald burn group with Pseudomonas aeruginosa.4.3 Changes of IL-1αlevels in serumThe serum level of IL-1αin three groups of scalded rats was higher than that before burned,and there was a significant difference（P<0.05）.There was a significant difference in IL-1αat 24 hours（P<0.05）,the scald burn aseptic group and the scald group with Pseudomonas aeruginosa were significantly higher than the SPF group.At the 3rd day,there was significant difference （P<0.05）,the scald burn aseptic group and the scald group with Pseudomonas aeruginosa were significantly higher than the SPF group.5 Changes of pathologyThe pathological sections showed that there were inflammatory cell infiltration,eosinophilic enhancement,and bleeding in the liver,kidney,and lung of the three groups of rats within one week after the scald,but there was no significant difference.There was no obvious inflammatory reaction in the pancreas.When the rats were stained with Pseudomonas aeruginosa for 24hours,lymphocytes were observed in the subcutaneous tissues and there were chronic inflammatory reactions.At 3 days,there was a purulent infection in the deep dermis,and a large number of inflammatory cells were seenaround the striated muscle.On the 7th day,there was slight blood vessel dilatation and congestion,and purulent infection was relieved.The skin of aseptic scald rats and SPF burned rats had mild to moderate inflammatory reactions within one week after scald.Conclusions:1 Sterile rats were obtained by purification of SPF rats from caesarean section,and they had fertility.2 Deep second degree scald model can be prepared by Innovative scalding method.3 After deep second degree scald,germ-free rats reacted fiercely,and serum inflammatory factors increased rapidly.Compared with germ-free rats,the inflammatory response of SPF rats was slower and more peaceful to a small area of deep second degree scald,maybe because they had stable immune system due to its self-contained bacterial flora.4 Compared with non P.aeruginosa infected aseptic group and SPF rats,pseudomonas aeruginosa infected deep second-degree scald germ-free rats has more shortened decrustation time and healing time.