With the approval of the Investigational Review Board and adherence to the Guide for the Care and Use of Laboratory Animals,23
all experiments utilized Sprague-Dawley postnatal day 7 (P7), 14, (P14) and 21 (P21) male and female rat pups (Charles River Laboratories, Wilmington, MA). They were housed in a room on a 12-h light/dark cycle with a free access to water. Rat pups were kept in cages with their littermates and mothers until weaning at postnatal age 21 and were housed with their littermates thereafter.
The experiments were conducted in a temperature-controlled acrylic container maintained at 36.7°C. In order to minimize handling of the pups, direct core temperature measurements were not obtained. However, similar conditions resulted in core body temperatures between 36.5 and 37.5°C.11
The rat pups were handled repeatedly by investigators to diminish effects resulting from stress-induced analgesia. The P7 and P14 rats were separated from their mothers during spinal injection and behavioral testing but were promptly returned for warmth and breast-feeding thereafter. The P21 rats were allowed free access to water and food at all times except during the spinal injection and behavioral testing. Rat pups for each age group were randomly assigned to spinal and general anesthesia as described in the sections to follow on “Intrathecal Injections of Bupivacaine” and “General Anesthesia Exposure”
Intrathecal Injections of Bupivacaine
The P7, P14, and P21 rats underwent baseline measurement of hind paw thermal withdrawal latencies (see Behavioral Measurements section below) immediately before spinal injection. The content of the spinal solutions were blinded to the investigator who performed the intrathecal injections (BY). With the animals in a prone position, the spinal solutions were injected intrathecally at the L4–L5 or L5–L6 level using a 100 μl syringe (26S gauge needle, model 801 RN; Hamilton Company, Bonaduz, Switzerland). Intrathecal placement of the needle tip was confirmed by observation of a tail flick.
Preliminary experiments indicated that awake P7 rats could be held un-anesthetized sufficiently still to perform these injections with high success rates, but that sufficient immobility was more difficult to achieve in awake P14 and P21 rats. For this reason, experiments in P14 and P21 rats were all performed after brief (less than 2 min) inhalation of isoflurane 2–4% (Baxter Healthcare Corporation, Deerfield, IL) vaporized in oxygen. The experiments in P7 rats were performed with separate groups of rats either awake or under brief isoflurane-anesthetized conditions, respectively. Doses were varied by maintaining a constant concentration of bupivacaine 7.5 mg/ml commercial solution (Hospira, Inc., Lake Forest, IL) and injecting varying volumes scaled to the rat pup’s body weight. Injected doses were 3.75 mg/kg as a low dose and 7.5 mg/kg as a high dose in all rats. Following spinal injections, animals were placed in a warm flat cage and thermal latencies were measured every ten minutes until full recovery and for a minimum of 40 min after injection. All rats receiving bupivacaine had successful blocks, as determined by signs of thermal blockade.
General Anesthesia Exposure
Groups of rats were exposed to either 1 h or 6 h of 1% inspired concentration of isoflurane in oxygen in a temperature-controlled chamber. Isoflurane was delivered using calibrated Matrx vaporizer (Matrx Medical Inc, Orchard Park, NY). Isoflurane and oxygen concentration were monitored by Capnomac Ultima Anesthesia Monitor (Datex-Ohmeda, Instrumentarium Corp., Helsinki, Finland) and animals were visually inspected for respiratory effort and skin color. While many previous publications on prolonged general anesthetic exposure in infant rats have not provided glucose supplementation during the anesthetic, there remains a concern that the neurologic effects of prolonged general anesthesia could be exacerbated by lack of substrate availability.7
In order to account for this possibility, separate groups of animals at each of the three ages receiving 6 h isoflurane exposures were treated with and without dextrose supplementation, respectively. Supplementation involved intraperitoneal injections of 5% dextrose, 0.05 ml, given hourly beginning after 1 h of anesthetic exposure.*
Methylene Blue Injections
Pilot experiments were performed to evaluate the spread of injectates in the spinal subarachnoid space. Using the same technique and age-specific injection volumes described above, rats received spinal injections of methylene blue 1 mg/ml solution. The extent of spread of the methylene blue was examined upon animal necropsy 10 min after intrathecal injections.
Behavioral Assessments for Sensory and Motor Blockade
Blockade of thermal nociception was assessed using a modified hot plate test, similar to what we have used previously in studies of peripheral nerve blockade.24,25
Hind paws were exposed in sequence (left then right) to a hot plate (model 39D hot plate analgesia meter; IITC Inc., Woodland Hills, CA) at 52°C for P7 and P14 pups and 56°C for P21 adolescents. The time (thermal withdrawal latency) until the rats lifted their paws was measured with a stopwatch. After 12 s, the tested paw was removed by the experimenter to avoid injury to the animal or the development of hyperalgesia. This test was repeated three times (with a 10-s pause between tests) for each rat at every time point. Thermal withdrawal latencies were measured every 10 min for at least 40 min after the intrathecal injection and until full recovery was observed.
Blockade of mechanical nociception was assessed by hind paw withdrawal to von Frey filaments. A series of filaments of increasing bending force was applied, and the force in grams of the filament that first evoked a withdrawal response was recorded. Mechanical withdrawal thresholds were recorded at baseline and every 10 min for at least for 40 min after the intrathecal injection and until full recovery was observed. Of note, for both the thermal and mechanical withdrawal tests, animals were observed for the possibility of exhibiting motor blockade without sensory blockade, i.e., by absence of lower limb movement accompanied by vocalization or signs of upper body escape responses. This was not observed for any animal.
Motor performance of the lower extremities was assessed by a qualitative score. For each leg, if there was no spontaneous or evoked movement, the contribution to the score was zero. If there was partial movement, the contribution was one, and if there was normal movement, the contribution to the score was two. Thus, in summing the values for both legs, the score could range from zero (complete blockade) to four (normal).
Motor behavior on Postnatal day 30
Motor impairment was assessed on the P30 rats that had undergone spinal bupivacaine or saline injections or 1 h or 6 h of 1% isoflurane general anesthesia interventions at postnatal day 7. These rats were introduced to a Dual species Economex Rotarod (Columbus Instruments, Columbus, OH) using a spindle rotating at 10 rotations per minute.11
Each rat was tested three times with 10 min intervals between each assessment. The maximal latency for each trial was 300 s before removal from the spindle. The average of the three assessments was used for data analysis. The investigator was blinded to the treatment groups.
Euthanasia and Perfusion
The animals were euthanized with intraperitoneal injection of sodium pentobarbital 100 mg/kg (Hospira, Inc.) and perfused transcardially with saline followed by 4% paraformaldehyde 6 h after the treatment periods ended. The spinal anesthesia groups (receiving bupivacaine or saline control), sham group, and 1-h isoflurane or 6-h isoflurane groups were perfused 6 h following initiation of the anesthetic intervention. After gentle dissection of the brain and spinal cord, the organs were stored overnight at 4°C in 4% paraformaldehyde and then transferred to a 30% sucrose solution at 4°C until sectioning, as detailed below. All samples were coded to allow for blinded histologic evaluation.
Blood Gas Analysis
A cohort of P7, P14, and P21 rats treated with spinal saline, spinal anesthesia at high and low dose, 1 h or 6 h (with or without dextrose as detailed above in the section on General Anesthesia Exposure) of 1% isoflurane general anesthesia underwent cardiac puncture 10 min after either spinal injection or the end of general anesthesia for the blood gas analysis. For all groups receiving spinal injections, appropriate presence or absence of blockade was confirmed by neurobehavioral criteria as per above. Rats were terminally anesthetized by intraperitoneal injection of sodium pentobarbital 100 mg/kg, the heart was exposed, and 0.3 ml of blood was taken by right ventricular cardiac puncture, for immediate blood gas analysis using a blood gas analyzer (pHOx Plus L, Nova Biomedical, Mississauga, Ontario, Canada).
Cleaved caspase-3 Immunohistochemistry
Fixed brains and spinal cords were placed in dry ice for 10–15 min and placed gently on a thin layer of Frozen Tissue Embedding Media (Fisher Diagnostic, Fairlawn, NJ) inside the cryostat box to create a block for sectioning. The brains and spinal cords were sectioned at 20 μm thickness using a cryostat (Leica CM3050S, Bannockburn, IL). Each slide contained 4 brain sections or 6 spinal cord sections. Coronal brain sections were obtained at a location that includes the lateral dorsal thalamus, ventrolateral division, and corresponds to an anteroposterior plane approximately 2 mm from bregma in the adult rat, similar to figure 26 in the Paxinos and Watson atlas.26
and lumbar sections of spinal cord were mounted onto Super Plus –coated glass slides (Fisher Scientific, Pittsburgh, PA). The slides were allowed to dry at room temperature for 24 h. Initially, the blocking solution (bovine-serum-albumin phosphate-buffered-saline Triton × 100 -Sodium Azide) was applied on the sections for half an hour at room temperature and then the blocking solution was removed from the sections to apply the primary rabbit anticleaved caspase-3 antiserum (1:2,500, Cell Signaling, Danvers, MA). Slides were then incubated overnight at room temperature. On the next day the sections were rinsed 3 times with phosphate-buffered saline, and then secondary antibody CY3 anti rabbit (1:100, Jackson Immunoresearch, West Grove, PA) was applied. Slides were incubated at room temperature for 2 h. After 2 h, secondary antibody was removed and the sections were washed three times in phosphate buffered saline and then given a final wash with 0.0 5M phosphate-buffered. Finally, the slides were allowed to dry before they were mounted over 3 drops of 90% glycerol with a cover slip. Microscopic evaluation was done using a BX-60 light microscope (Olympus, Southall, United Kingdom) at 20× magnification. A Hamamatsu digital camera C10600 ORCA-R2
(Photonic K.K., Hamamatsu City, Shizuoka, Japan) was used to take images of each section.
The investigator, blinded to the experimental cohort, counted the number of cleaved caspase-3 positive cells on one slide per animal either at the cortex in four brain sections or in six sections at the lumbar spinal cord. The number of cells counted reflected the sum of the cell counts per animal.
Histopathology of the spinal injection site
Animals from each age group were used for histopathologic evaluation of lumbar spinal cord. For P7, 10 animals were used, with five of the animals were treated with intrathecal injection of bupivacaine 3.75 mg/kg and five were injected with the same volume of saline. For P14 and P21, four animals each received intrathecal bupivacaine 3.75 mg/kg or saline, respectively. Rats received spinal injections as detailed above, and they were allowed to recover in their cages. All rats receiving bupivacaine (i.e., not saline controls) had successful blocks, as determined by signs of sensory and motor blockade per above. Seven days following injections, they were terminally anesthetized with Tribromoethanol (Avertin®, σ, St. Louis, MO) 2.5% 20 ml/kg, and underwent cardiac puncture for perfusion with cold phosphate buffered saline 30 ml, followed by 50 ml of cold fixative, containing 1.25% paraformaldehyde, 2.5% glutaraldehyde, 0.03% Picric Acid in 0.1M Cacodylate buffer. In brief, the histologic approach involved: fixation for 48 h, washing 2× in 0.1M cacodylate buffer, post fixation incubation for 48h in 1% OsO4 in 0.1M cacodylate buffer, washing - 2× 1h 0.1M cacodylate buffer, dehydrating in graded ethanol solutions: 50, 75, 95 100%(2×), suspension in Propylene Oxide: 1h - 2x, Propylene Oxide : araldite-ddsa (Epon) 1:1: overnight, embedding in epon, and then sectioning in 1 μm sections. Sections were mounted on slides, counterstained with Epoxy Tissue Stain (EMS, Hatfield, PA) and covered. Slides were coded and images have been taken at 10× and 60× magnifications with a Hamamatsu digital camera. The images were then examined by an expert in nerve structure and pathology (G.C.) who was blinded to the treatments.
For P14 and P21 rats, where there is sufficient maturation of tracts in the white matter of the lumbar spinal cord by the time of sacrifice 1 week after injections, a semiquantitative rank ordered scoring system was used, as per Estebe and Myers (2004).27
with the following 6 point Likert scale: zero was no pathology in any portion of the field, 1 was very few myelinated axons with any mild abnormality, 2 was slightly more myelinated axons with any abnormality than 1, but less than 10% abnormal, 3 referred to 10–20% abnormal myelinated axons, 4 referred to signs of moderate axonal degeneration, and 5 referred to clear signs of axonal degeneration. Since P7 rats, when sacrificed 1 week later at P14, have much less developed myelination and immature laminar organization of the spinal cord, a more qualitative interpretation was used by the examiner, who rated slides only as normal or not normal for age.
Thermal withdrawal latencies and von Frey mechanical withdrawal thresholds before and after bupivacaine or saline intrathecal injection was compared using a two-way mixed-model factorial analysis of variance (ANOVA) with age group (P7, P14, P21) and treatment (saline, bupivacaine 3.75 mg/kg, bupivacaine 7.5 mg/kg) as factors and F-tests were used to compare ages and treatments with a conservative post hoc Sidak-Bonferroni correction for multiple comparisons to protect against Type I errors. Blood gas parameters were compared between 6 treatment groups (spinal saline, spinal bupivacaine 3.75 mg/kg, spinal bupivacaine 7.5 mg/kg, isoflurane 1% 1h, isoflurane 1% 6h, isoflurane 1% 6h + dextrose supplementation) using analysis of variance (ANOVA) with Dunnett’s post hoc procedure to assess mean differences relative to the spinal saline (control) reference group. Hind leg motor response data are presented as median and interquartile range and compared between treatment groups for each age using the two-way nonparametric Friedman test using time as a repeated measures factor. Motor performance as measured by the Rotarod apparatus was compared between 4 groups (saline, bupivacaine 3.75 mg/kg, isoflurane 1% 1h, isoflurane 1% 6h) also by ANOVA with the F test used to ascertain differences with 95% confidence intervals constructed around the mean performance. Apoptosis as measured by caspase-3 activation using immunohistochemistry was compared between treatment groups in P7 rat pups by one-way ANOVA. Histologic injury scores were compared between spinal saline and spinal bupivacaine groups at ages P4 and P21 using Mann–Whitney U tests. Two-tailed values of P < 0.05 with correction as appropriate were considered statistically significant. Statistical analysis was performed with SPSS version 18.0 (SPSS Inc/IBM, Chicago, IL).