J .E. Irazuzta et al. Brain Research 881 2000 88 –97
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Cerebral edema with an increase in ICP and sudden Care and Use of Laboratory Animals published by the
herniation is the often fatal outcome of bacterial meningitis National Institutes of Health National Academy Press —
[26,31,46]. The raised ICP common to patients with revised 1996 and was performed with the approval of the
meningitis often occurs within 12 h of admission to the Wright State University Laboratory Animal Care and Use
hospital [27,46,47]. This time coincides with the increase Committee. Male white New Zealand rabbits 2.5–3 kg
in the inflammatory response generated by antibiotic were housed under conventional conditions. All surgical
therapy. This increase in the inflammatory response ex- procedures and the administration of bacteria were per-
acerbates blood–brain barrier dysfunction and cerebral formed under anesthesia. Anesthesia consisted of an
edema [53]. Recognizing the sequence of these events intramuscular administration of ketamine 30 mg kg and
offers a window of opportunity for studying interventions xylazine 3 mg kg followed by a continuous administra-
designed to attenuate the inflammatory response and treat tion of Isoflurane 1.5 and nitrous oxide 50.
or prevent secondary brain injury. Group B Streptococcus — type III GBS were cultured
To be successful interventions designed to attenuate the overnight in Todd–Hewitt broth and grown to logarithmic
inflammatory response, brain edema and increased ICP phase. The morning of the experiment the bacteria were
described in meningitis should modulate multiple points of washed and re-suspended in sterile saline. Meningitis was
the cascade of events that leads to increased brain damage. induced by inoculation into the cisterna magna of the
The neuroprotective role of hypothermia has been known animals with 0.4 ml of the GBS suspension after removal
for some time and hypothermia has been demonstrated to of 0.4 ml of cerebrospinal fluid CSF. Sham animals
have a beneficial effect at multiple points of brain injury received an inoculum containing sterile saline.
progression [40,50]. Moderate hypothermia reduces cyto- kine production, nitric oxide generation, leukocyte infiltra-
2.2. Randomization tion and the release of excitatory amino acids [18,22,63]. A
decrease in cerebral metabolism and preservation of energy Prior to randomization and sixteen hours after GBS
stores has been documented, as well as, a decrease in inoculation the severity of meningitis was determined by
cerebral edema [8,45,62]. In our laboratory we have two observers assigning a clinical severity score. Severity
demonstrated that the application of moderate hypothermia of illness was scored as follows: 05Normal activity, 15
decreases excitatory amino acid release and neuronal stress Reduced ambulation, 25inability to stand for .5 s, 35
in meningitis [29]. Moderate hypothermia has also been evidence of paralysis, 45actively seizing for more than 5
utilized in traumatic brain injury to control increased min or comatose. Animals were paired by severity of
intracranial pressure that has failed conventional therapy clinical symptoms and randomized to hypothermic 32–
[55]. 348C or normothermic 37–398C conditions. Animals
We postulate that the application of moderate hypo- were enrolled until there was a minimum of six survivors
thermia shortly after the administration of the antibiotics in each treatment group. Four control Sham animals
will attenuate the inflammatory response and increase in underwent the entire procedure but did not receive the
ICP that occurs in meningitis. Instituting hypothermia bacterial inoculum and were handled as animals in the
following antibiotic therapy has two characteristics that normothermic group.
make its study appealing. Hypothermia following anti- biotic therapy simulates a realistic clinical situation. The
2.3. Induction of hypothermia second advantage is that because inflammation increases
dramatically following antibiotic administration the use of Following randomization and instrumentation animals in
hypothermia as a therapeutic intervention will be coordi- the hypothermic group were rendered hypothermic by
nated with the time during which inflammation is in- covering their torso with a plastic bag containing ice.
creased. This study was performed in two parts. The first Stable rectal temperature conditions hypothermic, 32–
1 ]
was to evaluate the effects of moderate hypothermia on the 348C; normothermic, 37–398C were obtained within 1 h
2
integrity of the blood–brain barrier and inflammatory of initiating the antibiotic therapy. In a pilot study rectal
markers of brain injury. The second was to evaluate the and brain temperatures were equivalent in this model data
effects of hypothermia on intracranial pressure and the not shown. Instrumentation involved placement of a
maintenance of an adequate cerebral perfusion pressure in tracheotomy, a rectal probe, as well as, arterial and venous
animals with severe bacterial meningitis. catheters. Ventilatory support was initiated and titrated to
maintain a pH of .7.28, a PaCO of between 30 and 40
2
Torr and a PaO .100 Torr. Following instrumentation
2
2. Material and methods animals were given a 20 ml kg bolus of Hespan Excel ,
Irvine, CA and a continuous saline infusion at 20 ml h 2.1. Animal preparation
was started and continued for the duration of the study. Blood gasses were obtained every 2 h. If the PaCO was
2
This investigation conformed with the Guide for the within the target range, sodium bicarbonate was adminis-
90 J
tered at a dose of 0.5 mEq kg or 1 mEq kg for a pH,7.2 termined after enzymatic reduction of nitrate to nitrite
or pH,7.1 respectively. The temperature was monitored using nitrate reductase 670 mU ml with NADPH 160
with a rectal probe Physitemp 700 1 HT Physitemp mM at ambient temperature for 3 h. Following incubation
Instrument Inc., Clifton, NJ The temperature was main- an equal volume of the Griess reagent 1 sulfanilamide
tained in both groups with warming blankets Baxter K- and 0.1 naphthylethylenediamide in 5 phosphoric acid
MOD 100 Baxter Health Corporation, Deerfield, IL. was added and nitrite concentrations were measured
spectrophotometrically at 550 nm. 2.4. Protocol — the effects of hypothermia on
inflammation 2.7. Measurement of myeloperoxidase activity
In this component of the study animals received 0.4 ml Myeloperoxidase activity MPO in brain tissue was
5
of GBS 10 cfu ml. Sixteen hours after inoculation the determined as previously described with minor modifica-
animals received ceftriaxone Roche Laboratories 50 mg tion [6]. The mid and anterior portions of the left hemi-
kg IV, were instrumented and maintained under tempera- sphere were homogenized in a solution containing 50 mM
ture control conditions for 10 h. A dopamine infusion was Tris–HCl pH 7.4 at a ratio of 4 ml g of wet weight. An
initiated and titrated to maintain a mean arterial blood aliquot of 1 ml of the homogenate was added to 20 ml of 5
pressure of greater than 40 mmHg in all animals. The mM phosphate buffer pH 6 and centrifuged twice at 48C.
hypothermic animals were re-warmed prior to euthanasia. The supernatant was discarded, the pellet washed in
At the conclusion of the study anesthesia was briefly phosphate buffer and re-suspended in 0.5 hexa-decyl-
lightened to observe the presence of corneal reflexes and trimethyl-ammonium bromide. Three cycles of freezing
spontaneous respirations. The study animals then under- thawing in liquid nitrogen followed by sonication were
went a second cisternal tap for CSF collection and blood performed before incubation of the sample for 20 min at
samples were obtained by cardiac puncture. The animals 48C. An aliquot of the supernatant was mixed with a
were euthanized by an intracardiac administration of a solution of tetramethyl benzidine 3.9 mg ml in di-methyl
saturated solution of KCl formamide and 0.004 of H O . The rate of change in
2 2
Following euthanasia an autopsy was immediately per- absorbance was measured spectrophotometrically at 405
formed. The cranial vault was opened and its contents nm. Myeloperoxidase activity was defined as the quantity
extracted en masse. Following removal the right brain was of enzyme degrading 1 mmol of hydrogen peroxide per
isolated, stripped of the meninges, dissected on filter paper minute at 258C and expressed as units per gram of protein.
and frozen. In three animals from each group coronal The protein content of the homogenized brain was mea-
sections of the left brain were obtained fixed in formalde- sured spectrophotometrically by the Bradford method Bio
hyde, embedded in paraffin and cut for the preparation of Rad Sigma Diagnostics .
microscopic slides. The slides were stained with hemato- xylin and eosine according to standard techniques.
2.8. Protocol — the effects of hypothermia on cerebral perfusion and edema
2.5. Measurements of blood–CSF barrier function and
5
Infection We increased the bacterial load to 5310 cfu ml and the
dose of ceftriaxone to 100 mg kg in this component of the Blood and CSF specimens were centrifuged, the super-
study. Following the administration of antibiotics and natant collected and rapidly frozen for further analysis.
instrumentation animals in the hypothermic group were Alteration of blood–CSF barrier function was evaluated by
cooled as
previously described.
Animals were
in- measurement of protein and glucose content in CSF and
strumented as previously described with the addition of an
serum samples. Protein content was measured spectro- intracranial pressure monitor Camino , San Diego, CA
photometrically utilizing the Bradford method Bio Rad being placed by standard technique. After the surgical
Sigma Diagnostics . Glucose was measured spectro- procedures were performed nitrous oxide was discontinued
photometrically utilizing a commercially available assay and the isoflurane was decreased to 0.5. An infusion of
Glucose HK, Sigma Diagnostics . diazepam 0.7 mg kg h and fentanyl 35 mg kg h was
Bacterial presence was evaluated by culturing 10 ml of administered during the remainder of the study. Animals
both CSF and blood on 5 blood agar plate at 378C, were maintained hypothermic for 6 h.
followed by colony counts 24 h later. 2.9. Hemodynamic management
2.6. Measurement of NO production Maneuvers to maintain mean arterial pressure above 45
Nitric oxide was determined by measuring nitrate and mmHg were as follows. If the mean arterial pressure fell a
nitrite concentrations, the stable degradation products of second bolus of 20 ml of Hespan Excel , Irvine, CA was
NO, in CSF and serum samples [67]. Nitrite was de- given and repeated once more if the blood pressure did not
J .E. Irazuzta et al. Brain Research 881 2000 88 –97
91
improve. If volume resuscitation was inadequate a continu- were determined by unpaired t-test with the Bonferroni
ous dopamine infusion was initiated and titrated to main- correction for multiple comparisons. Regression analysis
tain an arterial blood pressure greater then 45 mmHg. was utilized when the association between continuous
During the last hours of the protocol the normal saline variables was sought. Determination of changes in heart
infusion was adjusted so that all animals received the same rate, blood pressure, intracranial pressure and cerebral
water load. Before euthanasia, the hypothermic animals perfusion pressure between experimental groups utilized
were re-warmed and the anesthesia was briefly lightened to repeated measures factorial ANOVA. Mann–Whitney U
observe the presence of corneal and paw reflexes as well as test was utilized for no-parametric data. Results were
