Mouse model of pneumococcal meningitis
A well-characterized mouse model of pneumococcal meningitis was used in this study . The model has been developed in C57BL/6 mice which are widely used for studies of cerebral infection and for studies on acquired hearing loss. C57BL/6 mice, however, carry the AHL locus for genetic hearing loss (Cdh23
) . All animals were of the same age (4-6 weeks), at a time point months before age-related hearing loss occurs in these mice . In brief, meningitis was induced by transcutaneous intracisternal injection of 15 μl of a bacterial suspension containing 106 colony forming units (cfu)/ml of S. pneumoniae (SP) D39 or placebo under short-term anaesthesia with enflurane. Mice were weighed, put into cages, allowed to wake up and fed with a standard diet and water ad libitum. Eighteen hours after infection, when all mice showed clinical signs of meningitis, intraperitoneal therapy with ceftriaxone was begun and continued for a total of 4 days. Furthermore, animals received adjunctive therapy or placebo (see experimental groups). Two weeks after infection, mice were deeply anaesthetized with ketamine and xylazine (SIGMA, St. Louis, MO, USA) and perfused transcardially with 15 ml of ice-cold phosphate buffered saline (PBS) containing 10 U/ml heparin. Temporal bones and brains were dissected, fixed in 4% formalin, decalcified in PBS containing 10% EDTA (SIGMA, St. Louis, MO, USA), and embedded in paraffin. All animal experiments were approved by the government of Upper Bavaria, Germany.
Experimental groups in the mouse model
The following groups were investigated: (i) mice intracisternally injected with 15 μl sterile phosphate-buffered saline (PBS) (CON; n = 8); (ii) mice intracisternally injected with SP, treated with 100 mg/kg ceftriaxone (Roche, Grenzach-Wyhlen, Germany) daily for 4 days and 0.5 ml isotonic saline every 2nd day for 2 weeks (PLC, n = 16); (iii) mice intracisternally injected with SP, treated with 100 mg/kg ceftriaxone daily for 4 days and 0.5 mg/kg dexamethasone (Merck, Darmstadt, Germany) every 8 h for a total of 4 days (DEX, n = 12); (iv) mice intracisternally injected with SP, treated with 100 mg/kg ceftriaxone daily for 4 days and 25 μg/kg Neurotrophin-3 (R&D Systems, Wiesbaden, Germany) every 2nd day for 2 weeks (NT-3, n = 11). Infected placebo-treated mice (PLC) were compared with uninfected mice (CON), showing successful infection and development of meningitis and meningitis-associated hearing loss. To detect a possible adjunctive treatment effect, animals who received adjunctive therapy with dexamethasone (DEX) or neurotrophin-3 (NT-3) were compared with placebo-treated mice (PLC) and with each other.
Clinical assessment of mice
Animals were investigated clinically before infection, 18 h, 42 h, and 2 weeks after infection. Clinical scores (CS) were determined as previously described reaching 0 points if there were no clinically noticeable signs of disease and 12 points if the animal died . In brief, the following criteria were assessed: (i) beam balancing, (ii) postural reflexes, (iii) piloerection, (iv) epileptic seizures, and (v) level of consciousness. For determination of explorative activity, each mouse was put in the middle of a 42 × 42 cm box divided into 9 squares and allowed to explore the box for two minutes. The number of squares which the mouse passed through within the two minutes time interval was counted.
Determination of hearing
Hearing was determined by auditory brain-stem responses (ABRs) at the end of the experiment. Mice were anaesthetized intraperitoneally with 100 mg/kg ketamine and 5 mg/kg xylazine. Needle electrodes were placed over each mastoid (negative pole), the vertex (positive pole), and the neck (reference). Impedances were controlled to be below 5 kΩ. Square-wave click impulses (duration: 100 ms; frequency: 20Hz) and tone bursts of 1 and 10 kHz (duration: 4 ms; frequency: 23.4 Hz) were delivered by earphones (E-A-RTONE3A, Aearo Company, Indianapolis, IN, USA). ABRs were amplified (x250.000), band-pass filtered (150-10,000Hz), and averaged (n = 1000) using a Neuroscreen Plus (Jaeger-Toennies, Freiburg, Germany). To determine the hearing threshold, we started with an impulse of 105 dB Sound Pressure Level (SPL) and reduced the intensity in 5 dB SPL steps. The lowest stimulus intensity that elicited visual ABRs was considered to be the hearing threshold. If a response could not be elicited at 105 dB SPL, stimulus intensities of up to 130 dB SPL were tested.
Histologic assessment of cochleae
For histological analysis, midmodiolar sections (thickness 7 μm) of mouse temporal bone were deparaffinized, rehydrated, and stained with Mayer's haematoxylin and eosin (H&E; MERCK, Darmstadt, Germany). Sections were digitized using an Olympus BX51 microscope (Olympus Optical, Hamburg, Germany), connected to a camera (Moticam 5000, Motic Deutschland GmbH, Wetzlar, Germany). Two sections of each cochlea were analyzed and the mean was calculated for statistics.
For determination of the spiral ganglion neuronal density in the cochlea, the area of each spiral ganglion was measured (Image Tool Version 3.00, University of Texas Health Science Center, San Antonio, TX, USA) and morphologically intact spiral ganglion neurons (criteria: round cell body containing a nucleus and homogenous cytoplasm) were counted within this area.
A regular cochlear finding 2 weeks after pneumococcal meningitis is fibrocytic occlusion of the scala tympani, indicating early labyrinthitis ossificans. The degree of occlusion was evaluated by measurement of the occluded area of the basal turn of the tympanic scala (Image Tool Version 3.00, University of Texas Health Science Center, San Antonio, TX, USA). The occluded area was expressed as a percentage to the total area of the basal scala tympani.
The integrity of the organ of Corti was evaluated as (i) existing and intact, or (ii) absent or respectively damaged. The integrity of the organ of Corti was defined as intact inner and outer hair cells as well as an intact architecture of supporting cells.
Immunohistochemical detection of neurotrophins and neurotrophin receptors
Cochleae were stained for NT-3 and BDNF as well as their receptors TrkB, TrkC (both inducing neuroprotective effects on neurons) and p75 (inducing apoptosis in neurons) using immunohistochemistry. Sections were deparaffinized and placed in a microwave for boiling in 10 mM citrate buffer (pH 6) for antigen retrieval. Endogenous peroxidases were blocked by incubation with 0.3% hydrogen peroxide in methanol (MERCK, Hohenbrunn, Germany). Sections were incubated with one of the following primary antibodies: (i) rabbit polyclonal anti-NT3 (1:500 in blocking solution, Alomone Labs Ltd, Jerusalem, Israel); (ii) rabbit polyclonal anti-BDNF (1:500 in blocking solution, Alomone Labs Ltd, Jerusalem, Israel); (iii) rabbit polyclonal anti-TrkB (1:500 in blocking solution, Novus Biologicals Inc., Littleton, CO, USA); (iv) goat monoclonal anti-mouse TrkC (1:200 in blocking solution, R&D Systems, Wiesbaden, Germany); (v) rabbit polyclonal anti-human p75 NTR (1:200 in blocking solution, Alomone Labs Ltd, Jerusalem, Israel) or (vi) blocking solution without primary antibody (negative control) in a humidified chamber overnight. Then, sections were incubated with secondary antibodies (biotinylated anti-rabbit IgG made in goat or anti-goat IgG made in horse according to the primary antibody used; both 1:200, Vector Labs, Burlingame, CA, USA). Binding of the secondary antibody was visualized with streptavidin horse-radish peroxidase (DAKO, Hamburg, Germany) and diaminobenzidine (Vector Labs, Burlingame, CA, USA), which yields a brown reaction product. Between all reaction steps, slides were rinsed with 0.1 M PBS (pH 7.4). Counterstaining was performed using Mayer's hematoxylin.
For assessment of immunohistochemical staining patterns, the most basal spiral ganglion was digitized. Pictures were blinded and printed on 20 × 30 cm color prints. Then, four reviewers (one professor of neuropathology, one assistant professor of neurology, and two residents of neurology with experience in neuropathology) independently grouped the printouts according to (i) homogeneity of the neuronal staining, (ii) intensity of the neuronal staining, and (iii) intensity of extracellular staining. A group was considered to be identified successfully if >80% of blinded slides were grouped together correctly.
Histologic assessment in the brain
For histological analysis, 7-μm-thick hippocampal sections of mouse brains were used. For assessment of general brain pathology, sections were stained with Mayer's haematoxylin and eosin (H&E), Prussian blue (MERCK, Darmstadt, Germany) (detection of iron as a marker for residues of parenchymal hemorrhages), glial fibrillary acidic protein (GFAP, detection of astrocyte activation (rabbit GFAP polyclonal, 1:3000, DAKO, Hamburg, Germany), and neurofilament and amyloid precursor protein (APP, detection of axonal injury, Neurofilament monoclonal, 1:500, DAKO; APP A4 monoclonal, 1:100, Chemicon, Billerica, MA, USA; secondary antibody: swine anti-rabbit biotinylated, concentration 1:150, DAKO, Hamburg, Germany, visualization with streptavidin-horse radish peroxidase and diaminobenzidine). Activated (GFAP-positive) astrocytes (appearing with a round nucleus and more than two GFAP-positive processes) were counted within predetermined areas (cortex and hippocampus).
All experimental procedures were performed in a blinded fashion. Data were analyzed with SYSTAT 9 (SPSS, Chicago, IL, USA) using a t-test for independent variables. P < 0.05 was considered significant. A Bonferroni correction was used to address the problem of multiple comparisons. Correlation analyses were performed according to Spearman. Data are displayed as mean ± standard deviation (SD).