Chronic Behavioral and Seizure Outcomes following Experimental Traumatic Brain Injury and ComorbidKlebsiella pneumoniaeLung Infection in Mice

Chronic Behavioral and Seizure Outcomes following Experimental Traumatic Brain Injury and Comorbid Klebsiella pneumoniae Lung Infection in Mice | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Chronic Behavioral and Seizure Outcomes following Experimental Traumatic Brain Injury and Comorbid Klebsiella pneumoniae Lung Infection in Mice Sarah S. J. Rewell , Ali Shad , Lingjun Chen , Erskine Chu , Jiping Wang , Ke Chen , Terence J. O’Brien , Jian Li , Pablo M. Casillas Espinosa , Bridgette D. Semple doi: https://doi.org/10.1101/2024.12.13.628278 Sarah S. J. Rewell 1 Department of Neuroscience, The School of Translational Medicine, Monash University , Melbourne, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ali Shad 1 Department of Neuroscience, The School of Translational Medicine, Monash University , Melbourne, VIC, Australia 2 Alfred Health , Melbourne, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lingjun Chen 1 Department of Neuroscience, The School of Translational Medicine, Monash University , Melbourne, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Erskine Chu 1 Department of Neuroscience, The School of Translational Medicine, Monash University , Melbourne, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jiping Wang 3 Department of Microbiology, Monash Biomedical Discovery Institute, Monash University , Clayton, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ke Chen 3 Department of Microbiology, Monash Biomedical Discovery Institute, Monash University , Clayton, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Terence J. O’Brien 1 Department of Neuroscience, The School of Translational Medicine, Monash University , Melbourne, VIC, Australia 2 Alfred Health , Melbourne, VIC, Australia 4 Department of Medicine (Royal Melbourne Hospital), University of Melbourne , Parkville, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jian Li 3 Department of Microbiology, Monash Biomedical Discovery Institute, Monash University , Clayton, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pablo M. Casillas Espinosa 1 Department of Neuroscience, The School of Translational Medicine, Monash University , Melbourne, VIC, Australia 2 Alfred Health , Melbourne, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bridgette D. Semple 1 Department of Neuroscience, The School of Translational Medicine, Monash University , Melbourne, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Bridgette.Semple{at}monash.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Traumatic brain injury (TBI) is a leading cause of long-term disability, and infections such as pneumonia represent a common and serious complication for TBI patients in the acute and subacute post-injury period. While the acute effects of infections have been documented, their long-term consequences on neurological and behavioral recovery as well as the potential precipitation of seizures after TBI remain unclear. This study aimed to investigate the chronic effects of Klebsiella pneumoniae infection following TBI, focusing on post-traumatic seizure development and neurobehavioral changes. Using a mouse model, we assessed the long-term effects of TBI and K. pneumoniae infection both in isolation and in combination. We found that, while infection with K. pneumoniae resulted in loss of body weight and increased mortality compared to vehicle-inoculated mice, there was no additional mortality in TBI animals. Further, although TBI alone induced chronic hyperactivity and reduced anxiety-like behaviors, K. pneumoniae lung infection had no lasting effect on these long-term outcomes. Thirdly, while TBI resulted in both spontaneous and evoked seizures long-term post-injury, early post-injury K. pneumoniae infection did not affect late onset seizure susceptibility. Together with recent findings on acute outcomes in this combined insult model of TBI and K. pneumoniae infection, this study suggests that K. pneumoniae does not significantly alter long-term neurobehavioral outcomes or the development of post-traumatic epilepsy. This research highlights the need to further explore the interplay between additional immune insults such as infection that may influence long-term recovery. BACKGROUND Traumatic brain injury (TBI) is a leading cause of long-term disability worldwide, with many survivors suffering from persistent cognitive, emotional, and physical impairments. Among the most significant acute and subacute complications following severe TBI are hospital-acquired infections, particularly pneumonia, which effects a substantial proportion of patients. 1 – 4 Pneumonia caused by Klebsiella pneumoniae , an opportunistic pathogen commonly associated with ventilator-associated infections, is of particular concern due to its role in exacerbating systemic inflammation and respiratory failure, and increasing the risk of mortality. 4 – 6 In TBI patients, immune dysregulation induced by the injury itself heightens vulnerability to such infections, which can worsen functional outcomes and complicate recovery. 7 – 9 Several recent studies, including from our group, have investigated the acute consequences of various infection models in the context of TBI and provided important insights into how bacterial infections exacerbate acute post-injury neuroimmune responses. 10 – 14 However, there remains a critical gap in understanding how pneumonia following TBI impacts long-term neurological and behavioral outcomes. Post-traumatic epilepsy (PTE) represents a debilitating long-term complication for survivors of moderate or severe TBI. 15 – 20 Defined as recurrent and unprovoked seizures that occur at least one week after TBI, 21 PTE can severely impact quality of life and is associated with an increased risk of cognitive decline, mood disorders, early-onset neurodegeneration and mortality. 22 – 26 Yet the precise mechanisms that drive the development of epileptogenesis after a brain injury remain elusive. Neuroinflammation, a well-known hallmark of TBI, is thought to play a role, and in this way, an additional immune challenge such as an infection is hypothesized to promote post-traumatic epileptogenesis. 27 , 28 To address this hypothesis, we recently conducted a retrospective cohort study examining a large trauma registry of adults with moderate to severe TBI. Infections were documented for approximately one quarter of TBI patients in the registry, with pneumonia being the most common presentation. By multivariate analysis to adjust for known risk factors, we found a solid association between hospital-acquired infections and the development of PTE at 2 years post-injury. 29 This finding suggests that hospital-acquired infections may contribute to the development of PTE, such that infections represent a modifiable risk factor. However, further exploration of this hypothesis in mouse models have to date failed to produce experimental evidence to support this theory. Specifically, we have evaluated the long-term consequences of peripherally-administered lipopolysaccharide (LPS), as an infection-like immune challenge, after experimental TBI in mice. In both pediatric and adult contexts, we reported that LPS induced a robust acute immune response yet did not exacerbate the long-term development of post-traumatic seizures. 10 , 30 While the LPS mouse model has some advantages as a well-established, predictable model of a systemic immune challenge, it fails to recapitulate many of the key features of a live bacterial infection in vivo . As such, it is pertinent that experimental models shift towards preferential use of live infectious agents, delivered via clinically-relevant routes, to more appropriately model the complex pathophysiological scenario of a hospital-acquired infection in an individual with severe TBI. 14 To address this, we recently established a new model of intratracheal inoculation of K . pneumoniae bacteria after experimental TBI in the mouse, and conducted detailed characterization of the acute consequences of this dual insult. 11 We observed that K. pneumoniae lung infection after TBI induced a robust yet transient inflammatory response, primarily restricted to the lungs but with some systemic effects, alongside exacerbated elevation of several pro-inflammatory genes such as Ccl2 in the brain of TBI + K. pneumoniae mice. However, the potential long-term consequences of these changes were not determined. The current study therefore sought to address this knowledge gap by investigating the chronic consequences of K. pneumoniae infection following experimental TBI, with a particular focus on the development of post-traumatic seizures and alterations in neurobehavioral outcomes. METHODS Experimental Timeline To determine the long-term effects of lung infection with a TBI, mice were subjected to a moderate-to-severe TBI model (or a sham control surgery), followed by intratracheal inoculation with Vehicle or K. pneumoniae bacterium at 4 days post-injury. The four experimental groups were Sham-Vehicle, Sham- Kp , TBI-Vehicle, and TBI- Kp . At approximately 4 months (16 weeks) post-injury, mice underwent extensive behavioral testing over a three-week period. At approximately 4-5 months post-injury, a recording electrode was implanted to allow for subsequent video-EEG monitoring for an average of 10 days per mouse. Finally, all mice received a sub-convulsive dose of pentylenetetrazol (PTZ) to evaluate evoked seizure responses, followed by tissue collection at 6 months post-injury ( Figure 1a ). Download figure Open in new tab Figure 1: Experimental timeline and acute outcomes. (a) Experimental timeline; (b) Body weight changes from the time of TBI or sham procedure (day -4) then up to 7 days post-inoculation with Kp or Vehicle. **p<0.01 indicates main effect of Kp from a three-way ANOVA. (c) Percent survival over the first two weeks post-inoculation. (d) Mortality rate in males. (e) Mortality rate in females. Animals and Ethics All animal experiments were conducted with approval from the Alfred Research Alliance Animal Ethics Committee (#P8032) and the Animal Care and Use Review Office (ACURO) of the US Department of Defense. These procedures adhered to the approved standards and the Australian Code for the Care and Use of Laboratory Animals as set by the National Health and Medical Research Council of Australia (NHMRC). Male and female C57Bl/6J mice were obtained from the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, and acclimated for one week before experiments began. The initial experiments (TBI surgeries and K. pneumoniae inoculations) were carried out in QC2 (microbiological containment) facilities at the Monash Research Animal Precinct in Clayton, Australia, before mice were transferred to the Precinct Animal Centre at the Alfred Hospital in Melbourne, for a 4-week quarantine period before behavior testing and video-EEG. The mice were housed in groups of same-sex littermates (2-6 per cage; with mixed experimental conditions per cage) in Optimice® individually-ventilated cages, maintained on a 12-hour light/dark cycle with continuous access to food and water. Controlled cortical impact (CCI) model of TBI Moderate-to-severe experimental traumatic brain injury (TBI) was induced in 10-12 week old mice using the controlled cortical impact (CCI) model, as previously described. 30 Briefly, anesthesia was initiated with 4% isoflurane in oxygen and maintained at 1.5% via a nose cone. Prior to surgery, all animals were given buprenorphine (0.05 mg/kg in saline; subcutaneously in the flank) and bupivacaine (1 mg/kg in saline; subcutaneously in the scalp) for pain relief, and 0.5 mL of 0.9% saline was administered at the end of the procedure for hydration. The mice were stabilized in a stereotaxic frame, and a 3.5 mm craniotomy was performed over the exposed left parietal bone. An electronic controlled cortical impactor device (eCCE-6.3; Custom Design and Fabrication Inc., Sandston, VA) was used to deliver an impact with a 3 mm rounded tip, at a speed of 4.5 m/s, to a depth of 1.7 mm for 150 ms. Sham animals underwent the same surgical procedure without the impact. After the CCI or sham surgery, the skin incision was sutured and an antiseptic solution applied. The animals were then allowed to recover in individual cages on a heat mat before being returned to their home cage. K. pneumoniae lung infection model Freeze-dried cultures of K. pneumoniae ATCC 15380 were obtained from In Vitro Technologies (Noble Park, VIC, Australia) and cultured on nutrient agar plates (Medium Preparation Unit, University of Melbourne, Victoria, Australia) at 37 °C overnight. For the initial culture, a large number of K. pneumoniae colonies were harvested and suspended in cation-adjusted Mueller-Hinton broth (CAMHB) for an additional 24-hour incubation at 37 °C with shaking at 180 rpm. Mid-logarithmic-phase cultures were then prepared by incubating in fresh CAMHB for 3 hours. The bacterial concentration (colony-forming units (CFU)/mL) was assessed by measuring the optical density (OD) at 600 nm and adjusted to the desired working concentration. Mice were randomized for inoculation with 10 6 CFU K. pneumoniae or vehicle on day 4 post-injury, as hospital-acquired infections are most common during the first week after a TBI. 31 , 32 A series of pilot studies were previously conducted to determine the optimal dose of K. pneumoniae via intratracheal inoculation to achieve an appropriate lung infection model in adult male and female mice. 11 For inoculation, mice were anesthetized with 2-4% inhaled isoflurane by an experienced technician and a MicroSprayer ® Aerosolizer (Penn-Century, Philadelphia, PA, USA) was used to administer 25 μL bacterial suspension or vehicle solution (diluted CAMHB) directly into the trachea. 33 Five-fold serial dilutions of both K. pneumoniae and vehicle samples were spiral plated and incubated overnight at 37 °C on nutrient agar plates to verify the CFU/mL in the administered inoculum, using ProtoCOL 3 software (Synbiosis, USA). Following recovery from the procedure, mice were closely monitored post-treatment for sickness behavior, general appearance, and weight loss over the time course. Weight loss ≥20% was a trigger for immediate humane euthanasia. Animal numbers are depicted in Table 1 . View this table: View inline View popup Download powerpoint Table 1: Experimental animal numbers. Facility Transfer The initial experiments (TBI surgeries and Kp inoculations) were carried out in QC2 facilities at the Monash Research Animal Precinct in Clayton, Australia, before mice were transferred to the Precinct Animal Centre at the Alfred Hospital in Melbourne for behavior testing and video-EEG monitoring. This experimental design was required due to the specialized facilities required for these procedures. Prior to leaving the Monash Clayton facility, for each of 4 cohorts of mixed-experimental groups, a health screen was performed at 6 weeks post-inoculation by sacrificing and testing a Swiss strain male mouse that was housed alongside the experimental mice and exposed to their bedding weekly. An additional health check was performed at the end of the four-week quarantine period upon arrival at the Alfred Hospital site. This involved the sampling of sera collected from 2-4 experimental animals that had previously had K. pneumoniae infection, via submandibular bleed performed by the animal facility technicians. Health screening was performed by Cerberus Sciences (Scoresby, VIC, Australia) via a standard panel. All cohorts passed the health screening. Once through the quarantine period, mice were transferred to the holding rooms in the Department of Neuroscience at Monash University, Alfred Hospital, and habituated for at least one week before behavior testing. Behavior Testing At approximately 16 weeks post-injury, a comprehensive battery of neurobehavioral tests was conducted to assess the chronic consequences of TBI and K. pneumoniae . Firstly, mice underwent an Open Field (OF) test, in a square arena for 10 min duration, to evaluate general locomotor activity, exploratory behavior and anxiety-like behavior. Anxiety-like behavior was additionally measured using the Elevated Plus Maze (EPM) during a 10 min period. In both tests, TopScan software was used to track activity and the time spent in the center versus periphery or open arms compared to the closed arms of the maze. 30 Gross sensorimotor performance was evaluated using the accelerating rotarod test over three consecutive days. Each day, mice completed three trials with a 30-minute rest period between trials. The rotarod device accelerated from 4 to 40 rpm over a 5-minute period, which was the maximum duration of the test. The average latency to fall from the rotarod was calculated for each mouse per day. 30 Social approach and social novelty preferences were assessed using the three-chamber test, which involved three consecutive 10-minute sessions conducted with a custom-built Perspex apparatus. The test proceeded through three stages: first, a habituation period (stage 1); then, the introduction of a same-sex stimulus mouse into one of the outer chambers (stage 2); and finally, the placement of a second, novel same-sex stimulus mouse into the opposite outer chamber (stage 3). TopScan software was used to monitor the amount of time the experimental mouse spent in each outer chamber. In stage 2, a preference for the chamber with the stimulus mouse over the empty chamber indicated social interest, while in stage 3, a preference for the novel mouse over the familiar mouse demonstrated social recognition or memory. 34 , 35 2 mice from the TBI-Vehicle group were unable to be analyzed from this test due to aberrant video tracking. Finally, the sucrose preference test was employed to identify potential depressive-like anhedonia in mice. Over a 5-day period, mice had access to two drinking bottles: one with filtered water and the other with a 1% sucrose solution. The positions of the bottles were switched halfway through the experiment. On the first and last day of the test, the volumes of liquid consumed from each bottle were measured, and a sucrose preference ratio was calculated by dividing the volume of sucrose solution consumed by the total volume of liquid consumed. 10 Video Electroencephalography (EEG) To evaluate chronic seizure activity, EEG electrodes were surgically implanted at 18-19 weeks post-injury. Under isoflurane anesthesia, epidural recording electrodes (E363/20/2.4/SPC ELEC W/SCREW SS, Plastics One Inc., USA) were carefully positioned: one ipsilateral and distal to the craniotomy, one contralateral to the craniotomy (2.5 mm to the right of the midline, -2.5 mm relative to Bregma), and two electrodes over the cerebellum for ground and reference. 36 An additional anchor screw (00-96 x 3/32, Plastics One Inc., USA) was placed over the left frontal region to reinforce the head cap. All screws were secured to the skull with SuperGlue (Bostik, Australia), and the electrodes were mounted into a pedestal head cap (MS363, Plastics One Inc., USA) and fixed with dental acrylate. The skin was sutured around the head cap, and the animals received subcutaneous pain relief (buprenorphine 0.05 mg/kg and bupivacaine 1 mg/kg) along with saline for hydration. Following electrode implantation, mice were housed individually for the remainder of the experiment. Phenotyping Post-Traumatic Epilepsy From 5 months post-TBI, continuous Video-EEG recordings were collected using a Grael EEG amplifier (Compumedics, Australia), accompanied by infrared video recordings. The EEG data was collected with a high-pass filter at 1 Hz and a low-pass filter at 70 Hz, at a sampling rate of 512 Hz, and digitized using Compumedics Profusion EEG software v 4.0. Recordings were obtained from both ipsilateral and contralateral electrodes, using common ground and reference signals. Each animal had a total of 8-12 days of video-EEG data recorded, which was analyzed using Assyst. 37 Seizures were identified by changes in the EEG pattern lasting more than 10 seconds, with an amplitude greater than three times the baseline, a repetitive and rhythmic discharge pattern, and variations in amplitude at the start and end of the seizure. 30 Potential seizure events identified by Assyst were reviewed by an experienced investigator (PMCE), who examined the EEG tracing blinded to the experimental group. Finally, identified seizures were confirmed following review of the video recording. Approximately 20% of video-EEG recordings were of insufficient quality to allow for accurate quantification, and were excluded. The presented results are from the remaining 80% of recordings. Pentylenetetrazol Seizure Susceptibility Challenge Immediately before tissue collection at 24 weeks post-TBI, a single intraperitoneal dose of 40 mg/kg pentylenetetrazol (PTZ) (P6500, Sigma, Australia) was administered to assess susceptibility to evoked seizures as an additional, indirect measure of PTE development. Behavioral responses to PTZ were observed over a 15-minute period. An experienced investigator (SSR), blinded to experimental group, reviewed the video recordings and rated the response to PTZ according to a modified 7-point Seizure Severity Score, where 0 indicates no response or normal activity, and 7 indicates status epilepticus leading to death. 30 , 38 , 39 Postmortem Lesion Assessment Mice were humanely euthanized via intraperitoneal injection of 160 mg/kg sodium pentobarbitone (Lethabarb®, Virbac, Australia), followed by transcardial perfusion with 4% paraformaldehyde (PFA) at a rate of 2 mL/min. The extracted brains were post-fixed overnight in 4% PFA, then transferred to 70% ethanol and sent to the Monash Histology Platform for paraffin processing and embedding (Monash University, Clayton, Australia). Seven μm coronal brain sections were sectioned and stained with cresyl violet and Luxol Fast Blue, as described previously, 11 , 30 to illustrate the extent of pathology. Statistical Analysis Statistical analysis was performed using GraphPad Prism v.9.4.1 (GraphPad Software Inc., San Diego, CA, USA), with significance defined as p<0.05. Two and three-way analyses of variance (ANOVA) were performed with Tukey’s post hoc test where appropriate. Data with 3 independent variables of time, injury, and infection, were assessed with a 3-way ANOVA. In most instances, both male and female mice are pooled per group, with open circle data points graphically denoting female animals. Potential sex differences were tested by 3-way ANOVA (factors of sex, injury, and infection) where appropriate, and only reported where significant sex differences were detected. Differences in mortality were tested using the Log-Rank (Mantel-Cox) test. Data are presented as mean ± SEM. RESULTS Impact of K. pneumoniae Infection on Body Weight and Mortality in TBI Mice We sought to test the hypothesis that lung infection with K. pneumoniae after a moderate-to-severe experimental TBI would exacerbate chronic behavioral and seizure outcomes. To determine this, four experimental groups (Sham-Vehicle, Sham- Kp , TBI-Vehicle, and TBI- Kp ) were compared across a range of outcome measures ( Figure 1a ). Body weights were monitored as an indicator of general health. When compared across the first week post-infection, a reduction in body weight due to K. pneumoniae inoculation is evident in Sham- Kp and TBI- Kp groups ( Figure 1b ) compared to vehicle-treated groups. Three-way ANOVA confirmed a main effect of time (F 4, 222 =28.36, p<0.0001), a main effect of Kp (F 1, 57 =9.36, p=0.0034), and a significant time x Kp interaction (F 1, 57 =9.06, p<0.0001). However, no effect of TBI alone was observed (F 1, 57 = 0.51, p=0.4776). K. pneumoniae inoculation resulted in acute symptoms associated with a lung infection, as expected as described previously in this paradigm. 11 A portion of mice were euthanized during the first week post-injury/infection, the majority between days 3-5 post-infection, due to excess weight loss ≥20% as per animal ethics guidelines ( Table 1 ). The total mortality rate for vehicle-treated mice was 5.7% (5.3% for males and 6.3% for females), and for Kp -infected mice it was 22% (17.2% for males and 27.6% for females). The combined insult of TBI and Kp infection appeared to increase mortality in male mice; however, this was not the case for female mice. There was no significant difference between mortality in Sham- Kp compared to TBI- Kp mice when analyzed over the first two weeks ( Figure 1c ; Log-Rank (Mantel-Cox) test, Chi-squared =0.0063, p=0.9366). Chronic Neurobehavioral Outcomes after TBI and K. pneumoniae Infection At approximately 16 weeks (4 months) post-TBI/Sham and K. pneumoniae infection, all experimental mice underwent a battery of neurobehavioral tests to assess long-term functional outcomes. Unless stated, no overt sex differences were observed. In the Open Field test, TBI mice showed an increase in total distance travelled compared to Sham groups (2-way ANOVA, F 1,55 =9.06, p=0.0039; Figure 2a ), indicating chronic hyperactivity as previously characterized in this model. 10 Similarly, TBI mice moved with a higher velocity compared to Sham mice (2-way ANOVA, F 1,55 =11.28, p=0.0014; Figure 2b ). However, Kp and Vehicle-treated groups performed similarly, and there were not TBI x infection interactions. Further, no effects of either TBI or infection were observed in terms of the proportion of time spent in the center of the arena (p>0.05). Download figure Open in new tab Figure 2: Neurobehavioral outcomes at approximately 4 months post-TBI / Kp infection. (a) Open Field (OF) test, total distance moved. (b) Velocity of movement in the OF test. (c) Time spent in the open arms of the Elevated Plus Maze (EPM). **p<0.01, ****p<0.0001, 2-way ANOVA main effect of TBI. (d) Rotarod, latency to fall. (e) Sucrose preference test. (f) Three-Chamber social approach test, Stage 1; (g) Stage 2; and (h) Stage 3. **p<0.01, ****p<0.0001, 2-way ANOVA main effect of TBI. In Stage 2, **p<0.01, ****p<0.0001 from post-hoc comparisons. Open circles = female; closed circles = male. Next, the Elevated Plus Maze was used as a measure of anxiety-like behavior ( Figure 2c ) . Here, TBI mice spent significantly more relative time in the open arms of the maze compared to Sham groups (2-way ANOVA, F 1,55 =23.79, p<0.0001), indicating a reduction in anxiety-like behavior chronically after injury as previously described. 10 Infection did not alter this response. In the Rotarod test ( Figure 2d ), while all experimental groups demonstrated improvement in performance over time (increased latency to fall from trials 1 to 3), no differences between the groups were observed nor were there any interactions between the factors of TBI, infection or time (3-way RM ANOVA, main effect of time p0.05). Finally, the Three-Chamber Social Approach Test was conducted. During habituation in Stage 1 ( Figure 2f ), as expected, no group differences were observed. In Stage 2 ( Figure 2g ), a significant main effect of chamber side was detected (3-way ANOVA, F 1,50 =87.71, p<0.0001). Tukey’s post-hoc analyses found that all groups showed a preference for the chamber containing the stimulus animal compared to the empty chamber side, indicating an intact preference for sociability. Lastly, in Stage 3 ( Figure 2h ), a main effect of chamber side was detected (F 1,50 =6.06, p=0.0173), as well as a chamber side x TBI interaction (F 1,50 =6.63, p=0.0131). Visually, it appears that Sham-Vehicle and Sham-Kp groups spent more time in the chamber containing the novel stimulus mouse compared to the familiar one, while both TBI groups spent roughly equivalent time in each chamber. However, Tukey’s post-hoc analyses failed to detect any specific differences between chamber sides for any of the experimental groups, rendering this stage difficult to interpret. Chronic Seizures After TBI and K. pneumoniae Infection Continuous Video-EEG recordings were obtained over an 8-12 day monitoring period per animal at approximately 5 months post-TBI/Sham, to evaluate whether early post-injury K. pneumoniae infection altered the development of PTE. Spontaneous electro-clinical seizure activity was observed in TBI-Vehicle and TBI- Kp mice ( Figure 3a-b ), but none of the sham-operated mice. All seizures were observed to be generalized in nature, commencing almost simultaneously in both hemispheres (ipsilateral and contralateral to the injury) and were typically Racine Class 3 or 4. A total of 7 animals (4 male and 3 female) exhibited at least one spontaneous seizure, and 40% of seizures occurred during the dark phase. All of the TBI-Vehicle mice had one seizure each during the recording period. Of the 3 TBI- Kp mice that had seizures, 2 had multiple seizures ( Table 2 ). In total, a comparable proportion of Vehicle and Kp-infected mice were observed to develop PTE: 21.1% of TBI-Vehicle mice (4/19) and 19.7% of TBI- Kp mice (3/16) (p>0.999, Fisher’s exact test; Figure 3c ). The average seizure duration was 67 sec (median 44 sec; range of 23-238 sec) ( Figure 3d ). Seizure duration was similar across the two experimental groups (t 8 =0.48, p=0.6421). Download figure Open in new tab Figure 3: Spontaneous and evoked seizure activity at 5-6 months post-TBI / Kp infection. Representative EEG tracings illustrate spontaneous seizure activity in a TBI-Vehicle (a) and TBI-Kp animal (b) over a 120 sec period. Ipsi = electrode positioned ipsilateral to the injury site; contra = contralateral to the injury site. Quantification (c) found a similar proportion of TBI-Vehicle and TBI-Kp mice exhibiting spontaneous seizures, of a comparable duration (d). Unpaired t-test, p>0.05. Finally, the evoked response to a PTZ challenge was evaluated (e), quantified as the Seizure Severity Score. **p<0.01, 2-way ANOVA main effect of TBI but no effect of Kp. n=12/group for Sham-Vehicle and Sham-Kp; 19 for TBI-Vehicle; 16 for TBI-Kp. Coronal brain sections (f) stained with cresyl violet and Luxol Fast Blue illustrate the typical extent of TBI damage observed following the CCI model at this chronic time point, in both TBI-Vehicle and TBI-Kp mice. Scale bar = 2 mm. View this table: View inline View popup Download powerpoint Table 2: Spontaneous Seizures Observed During Chronic Video-EEG Monitoring. Upon completion of continuous video-EEG recording, mice were administered 40 mg/kg i.p. PTZ and observed for a 15 min period. The behavioral response to PTZ was video-recorded then scored by a blinded investigator according to the modified Seizure Severity Score 10 , 36 ( Figure 3e ). 2-way ANOVA revealed that the average Seizure Severity Score in response to PTZ was higher in TBI compared to Sham mice (main effect of TBI, F 1,45 = 10.41, p=0.0023), while Kp had no effect (F 1, 45 = 0.38, p=0.5406). All mice were euthanized after the PTZ challenge and brain tissue collected for histology. A representative image of a TBI-Vehicle brain is depicted in Figure 3f , illustrating the typical pattern of unilateral cortical and hippocampal damage in the chronic phase as a result of the CCI model. DISCUSSION K. pneumoniae is a leading cause of ventilator-acquired pneumonia in critically-ill and immunocompromised individuals, and is common in patients with severe TBI. 31 , 40 – 43 After recently establishing a new mouse model of experimental TBI combined with a pulmonary K. pneumoniae infection, 11 in the current study we sought to investigate if the resolved infection had chronic consequences for TBI outcomes. In previous pilot studies, we determined 1 x 10 6 CFU of K. pneumoniae ATCC 15380 to be most appropriate for experimental use. 11 Here, we found that this dose induced considerable body weight and either spontaneous mortality or substantial weight loss triggering humane euthanasia for approximate 20% of infected mice (∼15% higher than mortality for Vehicle-treated mice). All mortality occurred within several days of infection, presumably due to pathology associated with the robust innate immune response triggered by K. pneumoniae inoculation, as demonstrated previously. 11 However, the combined insult of TBI plus infection did not exacerbate body weight loss, nor was there additional mortality observed in the TBI- Kp group compared to Sham- Kp mice. Of note, however, sex differences in mortality were observed. While females overall appeared to be more susceptible to K. pneumoniae infection, the combination of TBI+ Kp induced the highest mortality rate in male mice. With mixed reports of sex-specific responses to K. pneumoniae from experimental models, 11 , 44 – 46 potential sex-specific responses to infection warrant further investigation. Turning to long-term outcomes, we focused on neurobehavioral function and post-traumatic epilepsy. Of note, we have previously demonstrated in this model that most of the K. pneumoniae bacteria have been cleared from the lungs of surviving mice by 7 days post-infection, and certainly by a 28 day time point. 11 Here, we further confirmed that mice had resolved their infection prior to transfer between facilities for behavior testing and video-EEG, and no differences were observed between body weight trajectories of any experimental group after the initial two weeks (data not shown). We considered it plausible that prior K. pneumoniae infection, despite resolution, may nonetheless have long-term consequences for neurological function. For example, long-term deficits in exploratory locomotor behavior were reported in a mouse model of intranasal K. pneumoniae -induced pneumosepsis, associated with persistent brain inflammatory gene expression. 47 We have previously demonstrated that lung infection with K. pneumoniae alone induced acute expression of Tnfα in the cortex; while the combination of TBI plus K. pneumoniae resulted in elevated brain gene expression of pro-inflammatory cytokine Ccl2 and oxidative stress mediator Hmox1 . 11 However, at 4 months post-injury, we found that previously-infected K. pneumoniae mice were indistinguishable from Vehicle-treated mice across a range of tests for exploratory activity, sensorimotor function, anxiety-like behavior, social cognition, and pleasure-seeking behavior. TBI mice displayed chronic hyperactivity and reduced anxiety-like behavior as expected in this model of CCI. 10 , 30 The primary outcome of this study was the evaluation of post-traumatic seizures, based on recent epidemiological evidence of an association between hospital-acquired infection and the development of PTE by 2 years post-injury in patients. 48 From extended continuous video-EEG monitoring, we observed spontaneous seizure activity in approximately 20% of TBI mice, regardless of prior K. pneumoniae infection, and their seizures were of a similar nature and duration. In addition, both TBI groups showed a similar response to the PTZ challenge, an indirect measure of seizure susceptibility indicating epileptogenesis. This finding is in line with our previous experimental work using a similar experimental design but LPS as a mimic infectious agent, where LPS failed to have lasting effects on either neurobehavioral or seizure outcomes. 10 Together, these studies now suggested that either (a) there is not a robust or direct relationship between infection and these chronic outcomes; or (b) that the experimental models are still inadequate to replicate the clinical scenario. Of note, while live K. pneumoniae in this study was administered into the lungs (a clinically-relevant route), it was delivered as a single bolus which does not represent how a bacterial load expands over a time course in infected patients. 14 , 49 , 50 This may account for some of the mortality we observed, but may also influence the resulting immune response including brain-lung-immune interactions. Further, inherent differences between human and rodent immunity may render the mouse an insufficient model system for investigating the complex scenario of a critically-ill patient with TBI and infection. 14 Several strengths and limitations of the current study are worth noting. The study was carefully designed to dissect out specific effects of the combined insult of TBI plus K. pneumoniae compared to either TBI or infection alone, and is one of few studies to date to consider the long-term consequences of such a paradigm. Detailed seizure monitoring via continuous video-EEG is the gold-standard measure of PTE, and inclusion of both male and female mice allowed for consideration of typical biological variability as well as exploration of potential sex differences in responses to injury and infection. Conversely, the study was limited by lack of longitudinal analysis of seizure development, which could provide additional insight into seizure onset and progression over time. Use of a single infection model, time of administration relative to injury, and host strain/species, also limits the generalizability of the findings to other types of infections or pathogens. Finally, while this study focused on behavioral and seizure outcomes, we did not delve into the biological or molecular mechanisms that underlie these presentations. Future studies may benefit from the complementary inclusion of detailed histological or molecular analyses. CONCLUSION In conclusion, this study provides important insights into the potential long-term effects of K. pneumoniae infection following moderate-to-severe TBI. We found that a non-trivial K. pneumoniae lung infection, associated with ∼20% mortality, did not affect the chronic TBI-induced neurobehavioral deficits or PTE for the survivors. These findings suggest that while K. pneumoniae can worsen acute health parameters, its impact on long-term neurological outcomes post-TBI is limited once the infection has been resolved. Further research is needed to further explore potential sex-specific responses to infection, and consider how the timing of infection relative to injury may influence the host’s response. Overall, this work contributes to our growing understanding of how infections influence brain injury outcomes. Given the high incidence of both infections and seizures in TBI patients, and a growing concern over multidrug-resistant pathogens, such research is vital to the medical management of infections in TBI patients and support of optimal long-term outcomes. Funding This project was supported by an Epilepsy Research Program Idea Development Award (#W81XWH-19-ERP-IDA) from the US Department of Defense, awarded to BDS, JL and TOB. BDS was also supported by a Veski Near-Miss Grant. TOB is supported by a National Health and Medical Research Council of Australia (NHMRC) Investigator Grant (APP1176426). JL is supported by an NHMRC Principal Research Fellowship (APP1157909). PMCE is supported by the NHMRC (APP1087172 and APP2013629), a Monash Future Leader Fellowship (FLPF24-0237761657), a Medical Research Future Fund (MRFF) Stem Cell Therapy Missions Grant (MRF1201781), and the US Department of Defense USA Epilepsy Research Program (DoD ERP IDA, grant # EP200022, DoD ERPA RPA, grant# EP220067). Author Contributions Project conceptualization: BDS, TOB, JL. Data access and analysis: BDS, AS, SSR, LC, EC, KC, JW, PMCE. Manuscript draft: BDS. Manuscript edits: All authors. We confirm that this manuscript is consistent with the Journal’s and publisher’s position on issues involved in ethical publication. Ethics Approval All animal experiments were conducted following approval from the local Alfred Research Alliance Animal Ethics Committee (#P8032) as well as the Animal Care and Use Review Office (ACURO) from the Office of Research Protections, US Department of Defense, and carried out in accordance with these approved standards as well as the Australian Code for the Care and Use of Laboratory Animals as stipulated by the National Health and Medical Research Council of Australia (NHMRC). Conflicts of Interest The authors do not have any conflicts of interest to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements The authors would like to thank the Monash Animal Research Platform (Clayton and Alfred Precincts) for their assistance with the project. References 1. ↵ Jovanovic , B. , Milan , Z. , Markovic-Denic , L. , Djuric , O. , Radinovic , K. , Doklestic , K. , Velickovic , J. , Ivancevic , N. , Gregoric , P. , Pandurovic , M. , Bajec , D. and Bumbasirevic , V . ( 2015 ). Risk factors for ventilator-associated pneumonia in patients with severe traumatic brain injury in a Serbian trauma centre . International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 38 , 46 – 51 . OpenUrl PubMed 2. Kourbeti , I.S. , Vakis , A.F. , Papadakis , J.A. , Karabetsos , D.A. , Bertsias , G. , Filippou , M. , Ioannou , A. , Neophytou , C. , Anastasaki , M. and Samonis , G . ( 2012 ). Infections in traumatic brain injury patients . Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 18 , 359 – 364 . OpenUrl CrossRef 3. Li , Y. , Liu , C. , Xiao , W. , Song , T. and Wang , S . ( 2020 ). Incidence, Risk Factors, and Outcomes of Ventilator-Associated Pneumonia in Traumatic Brain Injury: A Meta-analysis . Neurocritical care 32 , 272 – 285 . OpenUrl CrossRef PubMed 4. ↵ Russo , E. , Antonini , M.V. , Sica , A. , Dell’Amore , C. , Martino , C. , Gamberini , E. , Bissoni , L. , Circelli , A. , Bolondi , G. , Santonastaso , D.P. , Cristini , F. , Raumer , L. , Catena , F. and Agnoletti , V . ( 2023 ). Infection-Related Ventilator-Associated Complications in Critically Ill Patients with Trauma: A Retrospective Analysis. Antibiotics (Basel , Switzerland ) 12 . 5. Clark , A. , Zelmanovich , R. , Vo , Q. , Martinez , M. , Nwafor , D.C. and Lucke-Wold , B . ( 2022 ). Inflammation and the role of infection: Complications and treatment options following neurotrauma . Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia 100 , 23 – 32 . OpenUrl PubMed 6. ↵ Fàbregas , N. and Torres , A . ( 2002 ). Pulmonary infection in the brain injured patient . Minerva anestesiologica 68 , 285 – 290 . OpenUrl PubMed 7. ↵ Dziedzic , T. , Slowik , A. and Szczudlik , A . ( 2004 ). Nosocomial infections and immunity: lesson from brain-injured patients. Critical care (London , England ) 8 , 266 – 270 . OpenUrl 8. Griffin , G.D . ( 2011 ). The injured brain: TBI, mTBI, the immune system, and infection: connecting the dots . Military medicine 176 , 364 – 368 . OpenUrl CrossRef PubMed 9. ↵ Sharma , R. , Shultz , S.R. , Robinson , M.J. , Belli , A. , Hibbs , M.L. , O’Brien , T.J. and Semple , B.D . ( 2019 ). Infections after a traumatic brain injury: the complex interplay between the immune and neurological systems . Brain, Behavior, and Immunity 79 , 63 – 74 . OpenUrl CrossRef PubMed 10. ↵ Rewell , S.S.J. , Shad , A. , Chen , L. , Macowan , M. , Chu , E. , Gandasasmita , N. , Casillas-Espinosa , P.M. , Li , J. , O’Brien , T.J. and Semple , B.D . ( 2024 ). A post-injury immune challenge with lipopolysaccharide following adult traumatic brain injury alters neuroinflammation and the gut microbiome acutely, but has little effect on chronic outcomes . bioRxiv , 2024.2009.2028.615631. 11. ↵ Shad , A. , Rewell , S.S.J. , Macowan , M. , Gandasasmita , N. , Wang , J. , Chen , K. , Marsland , B. , O’Brien , T.J. , Li , J. and Semple , B.D . ( 2024 ). Modelling lung infection with Klebsiella pneumoniae after murine traumatic brain injury . J Neuroinflammation 21 , 122 . OpenUrl CrossRef PubMed 12. Sharma , R. , Zamani , A. , Dill , L.K. , Sun , M. , Chu , E. , Robinson , M.J. , O’Brien , T.J. , Shultz , S.R. and Semple , B.D . ( 2021 ). A systemic immune challenge to model hospital-acquired infections independently regulates immune responses after pediatric traumatic brain injury . J Neuroinflammation 18 , 72 . OpenUrl CrossRef PubMed 13. Doran , S.J. , Henry , R.J. , Shirey , K.A. , Barrett , J.P. , Ritzel , R.M. , Lai , W. , Blanco , J.C. , Faden , A.I. , Vogel , S.N. and Loane , D.J . ( 2020 ). Early or Late Bacterial Lung Infection Increases Mortality After Traumatic Brain Injury in Male Mice and Chronically Impairs Monocyte Innate Immune Function . Crit Care Med . 14. ↵ Gandasasmita , N. , Li , J. , Loane , D.J. and Semple , B.D . ( 2023 ). Experimental Models of Hospital-Acquired Infections After Traumatic Brain Injury: Challenges and Opportunities . J Neurotrauma . 15. ↵ Christensen , J. , Pedersen , M.G. , Pedersen , C.B. , Sidenius , P. , Olsen , J. and Vestergaard , M . ( 2009 ). Long-term risk of epilepsy after traumatic brain injury in children and young adults: a population-based cohort study . Lancet 373 , 1105 – 1110 . OpenUrl CrossRef PubMed Web of Science 16. DeGrauw , X. , Thurman , D. , Xu , L. , Kancherla , V. and DeGrauw , T . ( 2018 ). Epidemiology of traumatic brain injury-associated epilepsy and early use of anti-epilepsy drugs: An analysis of insurance claims data, 2004-2014 . Epilepsy Res 146 , 41 – 49 . OpenUrl CrossRef PubMed 17. Ferguson , P.L. , Smith , G.M. , Wannamaker , B.B. , Thurman , D.J. , Pickelsimer , E.E. and Selassie , A.W . ( 2010 ). A population-based study of risk of epilepsy after hospitalization for traumatic brain injury . Epilepsia 51 , 891 – 898 . OpenUrl CrossRef PubMed Web of Science 18. Frey , L.C . ( 2003 ). Epidemiology of posttraumatic epilepsy: a critical review . Epilepsia 44 Suppl 10 , 11 – 17 . OpenUrl PubMed 19. Karlander , M. , Ljungqvist , J. and Zelano , J . ( 2021 ). Post-traumatic epilepsy in adults: a nationwide register-based study . J Neurol Neurosurg Psychiatry 92 , 617 – 621 . OpenUrl Abstract / FREE Full Text 20. ↵ Mariajoseph , F.P. , Chen , Z. , Sekhar , P. , Rewell , S.S. , O’Brien , T.J. , Antonic-Baker , A. and Semple , B.D . ( 2022 ). Incidence and risk factors of posttraumatic epilepsy following pediatric traumatic brain injury: A systematic review and meta-analysis . Epilepsia 63 , 2802 – 2812 . OpenUrl CrossRef PubMed 21. ↵ Scheffer , I.E. , Berkovic , S. , Capovilla , G. , Connolly , M.B. , French , J. , Guilhoto , L. , Hirsch , E. , Jain , S. , Mathern , G.W. , Moshe , S.L. , Nordli , D.R. , Perucca , E. , Tomson , T. , Wiebe , S. , Zhang , Y.H. and Zuberi , S.M . ( 2017 ). ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology . Epilepsia 58 , 512 – 521 . OpenUrl CrossRef PubMed 22. ↵ Kolakowsky-Hayner , S.A. , Wright , J. , Englander , J. , Duong , T. and Ladley-O’Brien , S . ( 2013 ). Impact of late post-traumatic seizures on physical health and functioning for individuals with brain injury within the community . Brain Inj 27 , 578 – 586 . OpenUrl CrossRef PubMed 23. Mazzini , L. , Cossa , F.M. , Angelino , E. , Campini , R. , Pastore , I. and Monaco , F . ( 2003 ). Posttraumatic epilepsy: neuroradiologic and neuropsychological assessment of long-term outcome . Epilepsia 44 , 569 – 574 . OpenUrl CrossRef PubMed Web of Science 24. Pingue , V. , Mele , C. and Nardone , A . ( 2021 ). Post-traumatic seizures and antiepileptic therapy as predictors of the functional outcome in patients with traumatic brain injury . Scientific reports 11 , 4708 . OpenUrl CrossRef PubMed 25. Semple , B.D. , Zamani , A. , Rayner , G. , Shultz , S.R. and Jones , N.C . ( 2019 ). Affective, neurocognitive and psychosocial disorders associated with traumatic brain injury and post-traumatic epilepsy . Neurobiol Dis 123 , 27 – 41 . OpenUrl CrossRef PubMed 26. ↵ Karlander , M. , Ljungqvist , J. , Sörbo , A. and Zelano , J . ( 2022 ). Risk and cause of death in post-traumatic epilepsy: a register-based retrospective cohort study . Journal of neurology 269 , 6014 – 6020 . OpenUrl CrossRef PubMed 27. ↵ Webster , K.M. , Sun , M. , Crack , P. , O’Brien , T.J. , Shultz , S.R. and Semple , B.D . ( 2017 ). Inflammation in epileptogenesis after traumatic brain injury . J Neuroinflammation 14 , 10 . OpenUrl CrossRef PubMed 28. ↵ Vezzani , A. , Fujinami , R.S. , White , H.S. , Preux , P.M. , Blumcke , I. , Sander , J.W. and Loscher , W . ( 2016 ). Infections, inflammation and epilepsy . Acta Neuropathol 131 , 211 – 234 . OpenUrl CrossRef PubMed 29. ↵ Chen , Z. , Laing , J. , Li , J. , O’Brien , T.J. , Gabbe , B.J. and Semple , B.D . ( 2024 ). Hospital-acquired infections as a risk factor for post-traumatic epilepsy: A registry-based cohort study . Epilepsia open 9 , 1333 – 1344 . OpenUrl CrossRef PubMed 30. ↵ Sharma , R. , Casillas-Espinosa , P.M. , Dill , L.K. , Rewell , S.S.J. , Hudson , M.R. , O’Brien , T.J. , Shultz , S.R. and Semple , B.D . ( 2022 ). Pediatric traumatic brain injury and a subsequent transient immune challenge independently influenced chronic outcomes in male mice . Brain Behav Immun 100 , 29 – 47 . OpenUrl CrossRef PubMed 31. ↵ Hamele , M. , Stockmann , C. , Cirulis , M. , Riva-Cambrin , J. , Metzger , R. , Bennett , T.D. and Bratton , S.L . ( 2016 ). Ventilator-Associated Pneumonia in Pediatric Traumatic Brain Injury . J Neurotrauma 33 , 832 – 839 . OpenUrl CrossRef PubMed 32. ↵ Lin , Y.W. , Zhou , Q. , Onufrak , N.J. , Wirth , V. , Chen , K. , Wang , J. , Forrest , A. , Chan , H.K. and Li , J . ( 2017 ). Aerosolized Polymyxin B for Treatment of Respiratory Tract Infections: Determination of Pharmacokinetic-Pharmacodynamic Indices for Aerosolized Polymyxin B against Pseudomonas aeruginosa in a Mouse Lung Infection Model . Antimicrobial agents and chemotherapy 61 . 33. ↵ Landersdorfer , C.B. , Wang , J. , Wirth , V. , Chen , K. , Kaye , K.S. , Tsuji , B.T. , Li , J. and Nation , R.L . ( 2018 ). Pharmacokinetics/pharmacodynamics of systemically administered polymyxin B against Klebsiella pneumoniae in mouse thigh and lung infection models . The Journal of antimicrobial chemotherapy 73 , 462 – 468 . OpenUrl CrossRef PubMed 34. ↵ Semple , B.D. , Canchola , S.A. and Noble-Haeusslein , L . ( 2012 ). Deficits in social behavior emerge during development after pediatric traumatic brain injury in mice . J Neurotrauma 29 , 2672 – 2683 . OpenUrl CrossRef PubMed 35. ↵ Dill , L.K. , Teymornejad , S. , Sharma , R. , Bozkurt , S. , Christensen , J. , Chu , E. , Rewell , S.S. , Shad , A. , Mychasiuk , R. and Semple , B.D . ( 2023 ). Modulating chronic outcomes after pediatric traumatic brain injury: Distinct effects of social and environmental enrichment . Exp Neurol , 114407 . 36. ↵ Semple , B.D. , O’Brien , T.J. , Gimlin , K. , Wright , D.K. , Kim , S.E. , Casillas-Espinosa , P.M. , Webster , K.M. , Petrou , S. and Noble-Haeusslein , L.J . ( 2017 ). Interleukin-1 Receptor in Seizure Susceptibility after Traumatic Injury to the Pediatric Brain . J Neurosci 37 , 7864 – 7877 . OpenUrl Abstract / FREE Full Text 37. ↵ Casillas-Espinosa , P.M. , Sargsyan , A. , Melkonian , D. and O’Brien , T.J . ( 2019 ). A universal automated tool for reliable detection of seizures in rodent models of acquired and genetic epilepsy . Epilepsia 60 , 783 – 791 . OpenUrl CrossRef PubMed 38. ↵ Cole , T.B. , Robbins , C.A. , Wenzel , H.J. , Schwartzkroin , P.A. and Palmiter , R.D . ( 2000 ). Seizures and neuronal damage in mice lacking vesicular zinc . Epilepsy Res 39 , 153 – 169 . OpenUrl CrossRef PubMed Web of Science 39. ↵ Sun , M. , Brady , R.D. , Wright , D.K. , Kim , H.A. , Zhang , S.R. , Sobey , C.G. , Johnstone , M.R. , O’Brien , T.J. , Semple , B.D. , McDonald , S.J. and Shultz , S.R . ( 2017 ). Treatment with an interleukin-1 receptor antagonist mitigates neuroinflammation and brain damage after polytrauma . Brain Behav Immun . 40. ↵ Gahagen , R.E. , Beardsley , A.L. , Maue , D.K. , Ackerman , L.L. , Rowan , C.M. and Friedman , M.L . ( 2023 ). Early-Onset Ventilator-Associated Pneumonia in Pediatric Severe Traumatic Brain Injury . Neurocritical care . 41. Kourbeti , I.S. , Vakis , A.F. , Ziakas , P. , Karabetsos , D. , Potolidis , E. , Christou , S. and Samonis , G . ( 2015 ). Infections in patients undergoing craniotomy: risk factors associated with post-craniotomy meningitis . J Neurosurg 122 , 1113 – 1119 . OpenUrl CrossRef PubMed 42. Zhang , X. , Zhou , H. , Shen , H. and Wang , M . ( 2022 ). Pulmonary infection in traumatic brain injury patients undergoing tracheostomy: predicators and nursing care . BMC pulmonary medicine 22 , 130 . OpenUrl CrossRef PubMed 43. ↵ Al Qasem , M.A. , Algarni , A.M. , Al Bshabshe , A. and Jiman-Fatani , A . ( 2023 ). The microbiological profile of isolates recovered from ICU patients with traumatic brain injuries at a tertiary care center, Southern Region, Saudi Arabia . Journal of infection and public health 16 , 1269 – 1275 . OpenUrl CrossRef 44. ↵ Durrani , F. , Phelps , D.S. , Weisz , J. , Silveyra , P. , Hu , S. , Mikerov , A.N. and Floros , J . ( 2012 ). Gonadal hormones and oxidative stress interaction differentially affects survival of male and female mice after lung Klebsiella pneumoniae infection . Experimental lung research 38 , 165 – 172 . OpenUrl CrossRef PubMed 45. Mikerov , A.N. , Cooper , T.K. , Wang , G. , Hu , S. , Umstead , T.M. , Phelps , D.S. and Floros , J . ( 2011 ). Histopathologic evaluation of lung and extrapulmonary tissues show sex differences in Klebsiella pneumoniae - infected mice under different exposure conditions . International journal of physiology, pathophysiology and pharmacology 3 , 176 – 190 . OpenUrl 46. ↵ Mikerov , A.N. , Gan , X. , Umstead , T.M. , Miller , L. , Chinchilli , V.M. , Phelps , D.S. and Floros , J . ( 2008 ). Sex differences in the impact of ozone on survival and alveolar macrophage function of mice after Klebsiella pneumoniae infection . Respir Res 9 , 24 . OpenUrl CrossRef PubMed 47. ↵ Denstaedt , S.J. , Spencer-Segal , J.L. , Newstead , M. , Laborc , K. , Zeng , X. , Standiford , T.J. and Singer , B.H . ( 2020 ). Persistent Neuroinflammation and Brain-Specific Immune Priming in a Novel Survival Model of Murine Pneumosepsis . Shock 54 , 78 – 86 . OpenUrl CrossRef PubMed 48. ↵ Laing , J. , Gabbe , B. , Chen , Z. , Perucca , P. , Kwan , P. and O’Brien , T.J . ( 2022 ). Risk Factors and Prognosis of Early Posttraumatic Seizures in Moderate to Severe Traumatic Brain Injury . JAMA neurology . 49. ↵ Bielen , K. , s Jongers , B. , Malhotra-Kumar , S. , Jorens , P.G. , Goossens , H. and Kumar-Singh , S. ( 2017 ). Animal models of hospital-acquired pneumonia: current practices and future perspectives . Annals of translational medicine 5 , 132 . OpenUrl CrossRef PubMed 50. ↵ Luna , C.M. , Sibila , O. , Agusti , C. and Torres , A . ( 2009 ). Animal models of ventilator-associated pneumonia . The European respiratory journal 33 , 182 – 188 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted December 17, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Chronic Behavioral and Seizure Outcomes following Experimental Traumatic Brain Injury and Comorbid Klebsiella pneumoniae Lung Infection in Mice Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Chronic Behavioral and Seizure Outcomes following Experimental Traumatic Brain Injury and Comorbid Klebsiella pneumoniae Lung Infection in Mice Sarah S. J. Rewell , Ali Shad , Lingjun Chen , Erskine Chu , Jiping Wang , Ke Chen , Terence J. O’Brien , Jian Li , Pablo M. Casillas Espinosa , Bridgette D. Semple bioRxiv 2024.12.13.628278; doi: https://doi.org/10.1101/2024.12.13.628278 Share This Article: Copy Citation Tools Chronic Behavioral and Seizure Outcomes following Experimental Traumatic Brain Injury and Comorbid Klebsiella pneumoniae Lung Infection in Mice Sarah S. J. Rewell , Ali Shad , Lingjun Chen , Erskine Chu , Jiping Wang , Ke Chen , Terence J. O’Brien , Jian Li , Pablo M. Casillas Espinosa , Bridgette D. Semple bioRxiv 2024.12.13.628278; doi: https://doi.org/10.1101/2024.12.13.628278 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17728) Bioengineering (13916) Bioinformatics (42037) Biophysics (21488) Cancer Biology (18636) Cell Biology (25552) Clinical Trials (138) Developmental Biology (13401) Ecology (19940) Epidemiology (2067) Evolutionary Biology (24367) Genetics (15621) Genomics (22545) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88744) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9834) Zoology (2272)