Lactobacillus Acidophilus Exerts Neuroprotective Effects In Mice With Severe Traumatic Brain Injury Via Gut-brain Axis And Its Revelation To Nursing

In traumatic brain injury（TBI）,secondary brain injury is the most essential phase that influences patients’survival and neurological functions.Thus,reducing damage from the secondary cascades is critical to improve functional outcomes of TBI patients.Recent years,more and more attentions have been focused on the role of gut-brain axis in TBI.The gut injuries caused by TBI is one of the initial factors leading to systemic inflammation.Improvement of gut functions is of great significance to inhibit excessive inflammation response and relieve secondary injury of neurological functions.Studies have found that gut microbiota plays a key role in regulating the gut-brain axis,which will affect the function of central nervous system.Probiotics are widely used to alter the composition of gut microbiota.Our previous animal studies showed that Lactobacillus acidophilus（LA）,an important strain of Lactobacillus,improves the morphology and contractility of ileum in severe TBI mice.In addition,the HE-staining results of brain tissue also presented that administration of LA can reduce brain edema and degeneration and necrosis of neuron cells of severe TBI mice,while this effect still need to be clarified and the specific mechanism involved in it is still unclear.In this study,we aim to validate whether LA has neuroprotective effects for TBI mice,explore the role of gut-brain axis in TBI and related mechanisms,and identify the importance of bowel care for patients’recovery.Our results can throw light on a new strategy to improve the dysfunctions of both gut and brain caused by TBI,and provide evidence for doctors and nurses to carry out treatments that based on the gut-brain axis theory.ObjectiveThe aim of this study was to investigate the neuroprotective effects of LA in TBI and elucidate underlying mechanisms,and provide new ideas for the treatments and nursing strategies of TBI patients regarding of the critical role of gut-brain axis in TBI.MethodThe C57BL/6 male mice（6-8w,21±3g）were randomly allocated to the following experimental groups before surgery:（1）sham+vehicle,（2）TBI+vehicle,and（3）TBI+?LA.The mice in TBI+vehicle and TBI+LA group were subjected to a severe TBI procedure.The sham+vehicle mice underwent craniotomy without brain injury.After injury,all mice were kept under standard conditions with access to food and water ad libitum.The mice in the TBI+LA group were orally gavaged with 1×10¹⁰ CFU of LA suspended in 0.5 ml sterile saline once daily until they were sacrificed,while the mice in the sham+vehicle and TBI+vehicle groups were gavaged with an equivalent volume of sterile saline.The first dose of LA was gavaged 4 h postoperation.1.Neurological score and rotarod test that assessing the sensorimotor functions were conducted with the same mice on the day before injury and 1,3,and 7 days post-TBI;the lesion volumes,and neuronal survival and degeneration in the perilesional cortex were tested by immunochemistry and Fluoro-Jade B staining.2.Three days post-injury,the dry-wet weight method,quantification of Evans blue dye extravasation and Western blot were used to measure the level of brain edema,permeability of blood brain barrier,and the protein expression of occludin in the injured cortex.3.Gliocyte aggregation,and levels of tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）in the injured cortex were measured by immunochemistry and immunofluorescence staining,and enzyme linked immunosorbent assay（Elisa）at 3 and 7days post-TBI.4.At 3 and/or 7 days after injury,we used HE staining to observe the structures of villus height and crypt depth in different intestine segments;Elisa kits were applied to measure serum D-lactate levels to assess the gut permeability;Western blot were conducted to test the expression of tight junction protein occludin in ileum.5.The colonization of LA and cecal microbiota composition were measured by Realtime PCR and 16S rRNA sequencing 3 days post-injury.6.At 3 and 7 days post-TBI,we used kits to measure levels of serum lipopolysaccharide（LPS）,TNF-αand IL-1β,and Realtime PCR to test the mRNA levels of toll-like receptor 4（Tlr4）and myeloid differentiation primary response gene 88（Myd88）in perilesional cortex.7.The neuron and glial cells of myenteric plexus were measured by immunofluorescence staining at 3 days after injury.Results1.LA improved neurological function in TBI mice（1）All groups of mice showed the same baseline in the behavioral tests pre-TBI,while at 1,3 and 7 days post-injury the sensorimotor functions were significantly impaired in TBI+vehicle mice.Administration of LA significantly improved the sensorimotor functions compared with TBI+vehicle group at 3 and 7 days after TBI.（2）The lesion volume and neuronal degeneration were significantly increased,and the number of neuron cells in injured cortex was decreased at 3 and 7 days after injury.Treatment with LA had no effects on the lesion volume,while the number of neuron cells was obviously increased and degenerative neuronal cells was reduced in the TBI+LA compared with TBI+vehicle mice 3 and 7 days post-TBI.（3）Three days following TBI,mice in the TBI+vehicle mice showed brain edema and elevated blood brain barrier permeability,as well as decreased expression of tight junction protein occludin.Administration of LA significantly reduced brain edema and permeability of blood brain barrier,and increased occludin protein expression compared with TBI+vehicle group at 3 days post-TBI.（4）On 3 and 7 days post-injury,glial cells gathered in the injured cortex,and levels of TNF-αand IL-1βwere significantly elevated.Number of total microglia,activated microglia and astrocytes,and concentrations of TNF-αand IL-1βin the injured cortex were significantly reduced after administration of LA 3 or 7 days after TBI.2.LA administration enhanced gut barrier function after TBI.（1）TBI significantly reduced the villus height and crypt depth in the duodenum,jejunum and ileum on days 3 and/or 7 after injury compared with sham+vehicle mice.Ileal villus height and crypt depth were higher in the TBI+LA mice than in the TBI+vehicle mice 3 and7 days postinjury.（2）At 3 and 7 days post-TBI,TBI significantly increased the intestinal permeability and decreased occludin protein expression.Treatment with LA for 3 or 7 days led to lower intestinal permeability than that observed in the TBI+vehicle group.The expression of occludin was significantly higher in the ileum of the TBI+LA mice than the TBI+vehicle group at the two time points.3.Effects of LA on gut-brain axis following TBI.（1）Administration of LA for 3 days significantly increased LA abundance in ileal mucosa compared with the TBI+vehicle group.（2）Three days after TBI,compared with the sham+vehicle group,the TBI+vehicle group had significantly decreased commensal richness（α-diversity）,and the gut microbiota composition in the TBI+vehicle mice was significantly altered.Abundances of some specific bacteria in different levels were also changed.Theα-diversity was increased in the TBI+LA group compared with the TBI+vehicle group.LA treatment reshaped the microbiota and the microbiota composition was more similar to that in the sham+vehicle group.Furthermore,abundances of some bacteria returned to normal level.（3）The levels of LPS and TNF-αwere elevated in the TBI+vehicle mice compared with the sham+vehicle group,and these increases were attenuated by LA treatment 3 and 7 days following TBI.（4）On day 3 or 7 postinjury,the mRNA expression of Tlr4 and its adaptor protein Myd88in the injured cortex of TBI+vehicle mice was significantly higher than that in the sham+vehicle group.The elevated mRNA expression of Tlr4 and Myd88 was attenuated in the TBI+LA group compared with the TBI+vehicle group 3 and 7 days after TBI.（5）The number of neuron cells and glial cells in the myenteric plexus was significantly reduced in TBI+vehicle group compared with sham+vehicle mice,while these reductions were attenuated by treatment with LA.Conclusions1.Administration of LA reduced the brain edema and neuronal loss in injured cortex,and improved the sensorimotor functions of TBI mice.LA might exert neuroprotective effects by inhibition of neuroinflammation in severe TBI.2.The structure of intestinal mucosa and intestinal barrier function were damaged after severe TBI,while treatment with LA can improve the morphology of ileum mucosa and decrease the intestinal permeability by enhancing the expression of tight junction protein occludin in TBI mice.3.LA can modulate the gut dysbiosis after TBI,decrease the intestinal barrier permeability and levels of serum LPS and TNF-α,and finally reduce the neurological disorders.Besides,the improvement of enteric nerve structure by LA may also contribute to LA’s neuroprotective effects via gut-brain axis.