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Developing a newborn rat model of meningitis without concomitant bacteremia by intraventricular injection of K1 (-) Escherichia coli

Neonatal meningitis caused by Escherichia coli results in high mortality and neurological disabilities, and the concomitant systemic bacteremia confounds its mortality and brain injury. This study developed an experimental model of neonatal meningitis without concomitant systemic bacteremia by determining the bacterial inoculum of K1 capsule-negative E. coli by intraventricular injection in newborn rats. Meningitis was induced by intraventricular intraventricular injection of 1 × 102 (low dose), 5 × 102 (medium dose), or 1 × 103 (high dose) colony forming units (CFU) of K1 (-) E. coli (EC5ME) in Sprague-Dawley rats at postnatal day 11. Ampicillin was started at postnatal day 12. Blood and cerebrospinal fluid (CSF) cultures were performed at 6 h, 1 day, and 6 days after inoculation. Brain magnetic resonance imaging (MRI) was performed at postnatal days 12 and 17. Survival was monitored, and brain tissues were obtained for histological and biochemical analyses at P12 and P17. Survival was inoculum dose-dependent, with lowest survival in high dose group (20x0025;) compared with medium (80%) or low (70%) dose group. CSF bacterial counts in low and medium dose group were significantly lower than that in high dose group at 6 h, but not at 24 h after inoculation. No bacteria were isolated from the blood throughout the experiment, or from the CSF at postnatal day 17. Brain MRI showed an inoculum dose-dependent increase in the extent of ventriculomegaly, cerebral infarct, extent of brain injury, and inflammatory responses. We developed a newborn rat model of bacterial meningitis without concomitant systemic bacteremia by intraventricular injection of K1 (-) E.coli.

survivors are not completely delineated. Therefore, a better understanding of the mechanism 58 of brain damage is necessary to prevent this neuronal injury, and consequently to reduce the 59 mortality and morbidities associated with neonatal bacterial meningitis. 60 Developing an appropriate animal model that could simulate clinical bacterial meningitis 61 in newborn infants would be essential to determine its pathogenesis, and also to test the 62 efficacy of newly developed adjuvant treatments in addition to the use of antibiotics. 63 Currently, several animal models of neonatal bacterial meningitis, including newborn piglets 64 (4), mice (5-7), rats (8, 9), or rabbits(10) are available, and meningitis was induced by various 65 routes including intraperitoneal (5, 11), intranasal (6), intravenous (5, 10, 12), or 66 intracisternal (7-10, 12) inoculation of bacteria. However, these animal models have certain 67 drawbacks, including small sample size, low infectivity, high mortality, and/or variable 68 extent of brain injury (11). Furthermore, concomitant bacteremia might aggravate the 69 meningitis-induced brain injury (9, 13, 14), thus increasing mortality (8,9,15). Therefore, in 70 the present study, we developed a newborn rat model of neonatal bacterial meningitis to 71 mimic the human clinical and neuropathological abnormalities, using 11-day-old newborn 4 72 Sprague-Dawley rats with titrated intraventricular inoculation of Escherichia coli, the most 73 common gram-negative pathogen of neonatal bacterial meningitis (3). We attempted to 74 determine the bacterial inoculum dose with maximal brain injury and minimal mortality by 75 using K1 capsule-negative E. coli to confine the infection to the central nervous system, 76 without concomitant systemic bacteremia (12, 16). We inoculated the bacteria 77 intraventricularly using a stereotaxic frame to simulate the neuropathological progression of 78 clinical neonatal bacterial meningitis, which begins with ventriculitis (17, 18). Brain injury 79 was monitored in vivo by brain magnetic resonance imaging (MRI) (19-22).

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Infecting organism 84 We used EC5ME, an un-encapsulated mutant of E. coli strain possessing the K1 capsular 85 polysaccharide C5 (serotype 018:K1:H7) (a kind gift from Professor Kwang Sik Kim, Johns 86 Hopkins University, MD, USA)(12, 16) to induce only bacterial meningitis, but not 87 secondary bacteremia, in this study. Bacteria were cultured overnight in brain heart infusion 88 broth, diluted in fresh medium, and grown for another 6 h to mid-logarithmic phase. The 89 culture was centrifuged at 5,000 ×g for 10 min, re-suspended in sterile normal saline to the 90 desired concentration, and used for intraventricular injection. The accuracy of the inoculum   The experiment began at P11, and continued through to P17. We assessed and monitored the 102 condition of rat pups on a daily basis regularly. To induce meningitis, newborn Sprague-

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Dawley rats (Orient Co, Seoul, Korea) were anesthetized using 2% isoflurane in oxygen 104 enriched air, and a total of 10 µl EC5ME inoculum in saline was slowly infused into the left 105 ventricle under stereotactic guidance (Digital Stereotaxic Instrument with Fine Drive, 106 MyNeurolab, St. Louis, MO, USA; coordinates: x = ± 0.5, y = ± 1.0, z = ± 2.5 mm relative to 107 the bregma) at P11. To determine the optimal inoculum dose with minimal mortality and 108 maximal brain injury, we tested three different inoculum doses of E. coli: A low inoculum 109 dose of 1 × 10 2 CFU EC5ME (LE), a medium inoculum dose of 5 × 10 2 CFU EC5ME (ME), 110 and a high inoculum dose of 1 × 10 3 CFU EC5ME (HE). For normal control group (NC), 111 equal volume of normal saline was given intraventricularly. After the procedure, the rat pups 112 were allowed to recover and returned to their dams, and there was no mortality associated 113 with the procedure. First, 10 rat pups for each group were allocated to assess the acute 114 pathophysiological changes, and the survivors were sacrificed at 24 h (P12) after bacterial 115 inoculation for histopathological assessment (n = 6, 5, 4 and 3 for the NC, LE, ME and HE 116 groups, respectively) and biochemical analyses (n = 4, 4, 4 and 3 for the NC, LE, ME and HE 117 groups, respectively). We also conducted the time course experiment in 10 animals for each 118 group to determine the survival rate until sacrifice of the survivors at P17 for 6 119 histopathological assessment (n = 5, 4, 4 and 2 for the NC, LE, ME and HE groups, 120 respectively) and biochemical analyses (n = 5, 3, 4 and 0 for the NC, LE, ME and HE groups, 121 respectively). Intraperitoneal injection of ampicillin (200 mg/kg/day) was started 6 h after 122 bacterial inoculation, and continued for 3 days until P13. CSF was obtained to determine the 123 bacterial titer at 6 h, 24 h, and 6 days (P17) after bacterial inoculation. Brain MRI was 124 performed at P12 and P17. All experimental procedures generating pain were performed 125 under the isoflurane inhaled anesthesia to reduce pain. All animals were daily monitored and 126 we assessed mortality. Every cause of death was not associated with experimental procedures 127 and related to disease condition. At P12 and P17, survived animals were euthanized by 128 isoflurane and sacrificed by cervical vertebra dislocation and whole brain tissue and CSF 129 samples were obtained.   The brain MRI was performed while the rats were kept in an anesthetized state by the 144 administration of 1.5-2% isoflurane in oxygen-enriched air using a facemask. All MRI 145 examinations were performed using a 7.0-tesla MRI System (Bruker-Biospin, Fällanden, 146 Switzerland) prepared with a 20-cm gradient set capable of providing a rising time of 400 147 mTm-1. The MR images were acquired with 1.0-mm slice thickness, and a total of 12 slices 148 were acquired. Brain MRI was performed at P12 (n = 10, 9, 8 and 6 in the NC, LE, ME and 149 HE groups, respectively) and at P17 (n = 11, 7, 8 and 2 in the NC, LE, ME and HE groups, 150 respectively). After the MRI exams, the rat pups were allowed to recover and were returned 151 to their dams. 154 All MR images were analyzed using Image J software (National Institutes of Health). The 155 lesion was well identified by the hyperintense areas in DWI at P12 and by the hyperintense 156 areas in T2-weighted imaging at P17. The ratio of the infarcted region in the cortex to the 157 whole brain volume was calculated as a parameter of brain injury. The ventriculomegaly 158 volume ratio was also calculated for each pup.    survival rate up to postnatal day (P)17 of 20% for the high inoculum dose (HE), and 70% and 220 80% for LE and ME doses, respectively (Fig 2A). While survival rate up to P17 in the HE 221 group was significantly lower compared to that in the no inoculum control (NC), the survival 222 rate of the LE and ME groups was not significantly reduced compared with the NC group.

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While birth body and brain weight in each study group was not significantly different 224 between the study groups; the body weight gain at P17 in the LE, ME, and HE groups was 225 significantly lower, the brain weight gain in the ME and HE groups was significantly lower, 226 and the brain/body weight ratio in the ME and HE groups was significantly higher compared 227 with the those in the NC group. The least body and brain weight gain, and the highest 228 brain/body ratio, were observed in the HE group compared with those in the LE and ME 229 groups (Fig 2B-D). are presented as the mean ± standard error of the mean (SEM). * P < 0.05 compared with the 238 NC group, # P < 0.05 compared with the LE group, $ P < 0.05 compared with the ME group.  Thereafter, the bacterial counts in the CSF of all study groups increased significantly 247 compared with that at 6 h, and there were no significant inter-group differences at 24 h after 248 the induction of meningitis (Fig 3). No bacterial growth in the CSF was detected all study 249 groups at 6 days after the induction of meningitis.  To assess the extent of meningitis-induced brain infarction and hydrocephalus, in vivo 259 brain MRI scans were taken. The degree of the brain infarct in the ipsilateral cortex and the 260 dilatation of the ventricle to whole brain as evidenced by the hyperintense areas in the 12 261 diffusion-weighted MRI performed at P12 and by T2-weighted MRI performed at P17 were 262 measured.

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The brain infarct volume ratios at P12 and P17 were bacterial inoculum dose-dependently 264 increased, showing the highest ratio in the HE group, and a seemingly increased ratio in the 265 ME group compared with that in the LE group that did not reach statistical significance (Fig   266   4). The ventriculomegaly volume ratios at P12 were bacterial inoculum dose-dependently 267 increased, showing the highest increase in the HE group compared with that in the LE and 268 ME groups. In addition, although the absolute extent of ventriculomegaly was significantly 269 reduced compared with P12, the ventriculomegaly volume ratios at P17 were also bacterial 270 inoculum dose-dependently increased, showing the highest increase in the HE group 271 compared with that in the LE and ME groups (Fig 4).    compared with those in the LE group (Fig 6). Although the brain homogenates of the HE 310 group were not available for measurements because of their high mortality at P17, and the 311 absolute levels of the inflammatory cytokines were significantly reduced compared with P12, 312 the inflammatory cytokines were bacterial inoculum dose-dependently increased, showing 313 significantly higher levels in the ME group compared with those in the LE group.  lie in the great variability between the species, the inoculation methods, and the age of the 332 animal models (11). In this study, we used P11 rats as an animal model of neonatal 15 333 meningitis because the rat brain at P11 is comparable in terms of maturation to the human 334 brain at birth (23). The larger size of rat pups compared with mice enables easier surgical 335 manipulation at an earlier age, and a larger amount of brain tissues obtained at harvest.  In this study, E. coli was used to induce meningitis, because it is the most frequent 344 gram-negative pathogen of neonatal bacterial meningitis (3). Although brain injury primarily 345 results from local meningeal infection, concomitant systemic bacteremia might aggravate the 346 meningitis-induced disease severity, brain injury, and mortality (9, 13-15). This discordance 347 between disease severity and brain injury means that a poorer outcome does not necessarily 348 lead to increased brain injury (15,19). In addition, neuroprotection might not be associated

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In the present study, we tested three different doses of K1 (-) E. coli (EC5ME) for the 372 induction of meningitis to determine the optimal inoculum dose with minimal mortality and 373 maximal brain injury; 1 × 10 2 CFU for the LE group, 5 × 10 2 CFU for the ME group, and 1 × 374 10 3 CFUs for the HE group. Survival rates, body and brain weight gain, the extent of 375 inflammatory responses and brain injury correlated significantly with the inoculum dose used 376 to induce meningitis, showing highest mortality, extent of inflammatory responses, and brain 377 injury, and the least body and brain weight gain. We also observed higher inflammatory 378 responses and the least extent of brain injury in the ME and LE groups, respectively. The 379 mortality rate was positively correlated with the inoculum dose and the extent of 380 inflammatory responses and brain injury. As blood culture was negative throughout the 17 381 experiment, the inoculum dose-dependent increase in mortality, inflammatory responses, and 382 brain injury solely reflects the virulence of EC5ME meningitis, without the confounding 383 effects of the concomitant systemic bacteremia. Overall, these findings suggest that ME (5 × 384 10 2 CFU) of EC5ME might be optimal inoculum dose to induce neonatal meningitis.

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Because bacterial meningitis induces high mortality in newborn infants, the design of 386 animal study was also driven to target for severe, end-stage models. From an ethics 387 viewpoint, this use contradicts views that death as an endpoint is unacceptable. However, the