Keywords

Introduction

No matter how carefully a neurosurgical procedure is performed, it is intrinsically linked to postoperative deficits resulting in delayed healing caused by direct trauma, hemorrhage, and brain edema, termed surgical brain injury (SBI) [1, 3, 7, 11]. Cerebral edema and hemorrhage are major postoperative complications of SBI and are major contributors to patient morbidity, resulting in increased postoperative care.

Although there are several interventions utilized to minimize bleeding and damage to healthy tissue surrounding the retraction site, there is still significant burden placed on the US health-care system for postoperative recovery of neurosurgical patients [2]. Although novel therapies are being developed in preclinical models, translation of these therapies is limited, possibly because of the inadequate use of neurobehavioral testing [16, 21].

The SBI rodent model [11] mimics clinical SBI pathophysiology, including intraoperative hemorrhage, brain edema, neuroinflammation, and postoperative hematoma [10, 11]. Although numerous behavioral tests are available to assess functional recovery after injury [14, 15, 17, 18], many of them have not been examined for the SBI rodent model. Furthermore, the choice of behavioral test depends on the neurological disease of interest; SBI significantly impairs the sensorimotor function.

The most common sensorimotor tests for rodents are the composite Garcia neuroscore and beam walking test. Although these tests were first utilized for assessing function in rodents after ischemic brain injury [21], they have also been utilized in the rat SBI model [11]. However, a variety of alternative sensorimotor tests exist and have not been the focus of functional assessment after SBI [4, 12]. Furthermore, no publication has yet examined whether a correlation between brain water content and functional deficits exists after SBI in rats. Thus, herein we examine the correlation between neurological function and brain water content in rats 24 h after SBI.

Materials and Methods

All experiments were approved by the Institutional Animal Care and Use Committee at Loma Linda University. Adult male Sprague-Dawley (SD) rats (290–320 g) were used in all experiments.

Surgical Brain Injury Model

SBI was induced in male SD rats via partial unilateral frontal lobectomy as previously described [11]. Briefly, animals were anesthetized with 3 % isoflurane in O2 mixed with medical gas (4.5 L/min initial, 2.5 L/min sustained). Anesthetized animals were placed into a standard rodent stereotactic frame, a midline skin incision was made, and a right-sided craniectomy (5 × 5 mm) was performed (1 mm posterior to the bregma and 2 mm lateral to midline). The right frontal lobe was sharply dissected to the base of the brain. Hemostasis was achieved using a combination of electrocautery, direct pressure with hemostatic dressings, and normal saline irrigation. Once intraoperative hemorrhage stopped, the cranial window was sealed with bone wax, the skin was sutured, and animals were allowed to recover. Animals received subcutaneous injections of 0.9 % normal saline (1 mL) and buprenorphine (0.03 mg/kg).

Neurobehavior Testing

Twenty-four hours after injury, neurobehavior was assessed using the composite Garcia neuroscore (n = 10), beam walking test (n = 10), corner turn test (n = 10), and beam balance test (n = 35) as previously described [5, 8, 13, 21].

Briefly, composite Garcia neuroscore [5] consists of seven independent subtests (spontaneous activity, axial sensation, vibrissae proprioception, limb movement symmetry, lateral turning, forelimb walking, and climbing), each scored between 0 (completely impaired function) and 3 (retained complete function). The neuroscore for sham versus SBI animals was analyzed using the Mann–Whitney test and presented as mean ± standard error of the mean (SEM).

Assessment of beam walking ability [21] was conducted by placing rats midway on a horizontal round beam (90 × 2 cm) connected to two platforms at the beam ends. Rats were given 40 s to move along the beam. Scoring was as follows: 0 (worst performance) if animals fell off the beam in less than 40 s, 1 if animals stayed on the beam for at least 40 s, 2 if animals moved less than halfway to a platform (in less than 40 s) and stayed on the beam at least 25 s, 3 if animals moved more than halfway to a platform and stayed on the beam at least 25 s, 4 if animals reached a platform in less than 40 s, 5 if animals reached a platform in less than 25 s. The beam walking score for sham versus SBI animals was analyzed using the Mann–Whitney test and presented as the mean ± SEM.

The corner turn test [9] examined preference for left versus right turning. Rats approached a 30° corner, and to exit, animals had to turn around along the corner, turning to the left or right. The choice of turning side was recorded for 20 trials with at least 30 s between each trial. Corner turn test score was calculated ((as number of left turns)/(total turns)) × 100.

The beam balance test [6] examines proprioception and balance using a horizontal Plexiglas beam (2 × 80 cm, 25 cm tall). The animals were placed midway on the beam, and with an innate tendency to avoid falling, animals walked along the beam. A total of 3 min was given for the beam balance test. The distance traveled during the 3 min was scored in 1-cm ticks.

After neurological testing, animals were sacrificed for right frontal lobe brain water content evaluation using the wet-dry method [20]. Under deep anesthesia, animals were decapitated and brain samples quickly removed. Dissected brains were separated into frontal and parietal lobes for the ipsilateral (right) and contralateral (left) hemispheres, then weighed for wet weights. The samples were dried at 100 °C for 24 h before measuring the dry weights. Brain water content was calculated as ((wet weight − dry weight)/(wet weight)) × 100. Brain water content was analyzed using unpaired t-test and presented as the mean ± SEM.

All behavioral data are presented as the individual data points and corresponding brain water content. Correlation between brain water content and neurological deficits was analyzed with the Spearman’s rank correlation coefficients (ρ) and best-fit linear regressions were determined.

Results

The brain water content of animals subjected to SBI (82.57 ± 0.313 %) is significantly elevated compared with sham-operated animals (80.27 ± 0.178 %) (p < 0.05, Fig. 1a).

Fig. 1
figure 1

Brain water content and neurobehavior differences after SBI. (a) Brain water content (sham n = 7, SBI n = 12). (b) Composite Garcia neuroscore (sham n = 7, SBI n = 12). (c) Beam walking score (sham n = 7, SBI n = 12) *p < 0.05 vs Sham

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Neurobehavior

Twenty-four hours after SBI, before euthanasia, animals were subjected to four neurobehavioral tests: composite Garcia neuroscore (neuroscore), beam walking test, corner turn test, and beam balance test.

Composite Garcia Neuroscore

The neuroscore of injured animals (15.8 ± 0.52) was statistically lower than that of sham animals (19.71 ± 0.18) (p < 0.05, Fig. 1b) 24 h after SBI. However, while statistically lower than SBI-injured animals, the neuroscore did not significantly correlate with the brain water content of the right-frontal lobe 24 h post-ictus (ρ = −0.02, p > 0.05, Fig. 2a).

Fig. 2
figure 2

Neurobehavior correlation with brain water content in animals subjected to SBI. (a) Composite Garcia neuroscore (n = 10). (b) Beam walking score (n = 10). (c) Corner turn test (n = 10). (d) Beam balance test (n = 35). All correlations were tested for correlation using Spearman’s rank correlation coefficient (ρ) and statistical significance (p-value)

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Beam Walking

Beam walking score for SBI-injured animals (1.4 ± 0.36) was significantly lower than sham animals (4.3 ± 0.36) (p < 0.05, Fig. 1c). Yet, no significant correlation was observed between the beam walking score and right-frontal lobe brain water content 24 h after SBI (ρ = −0.33, p > 0.05, Fig. 2b).

Corner Turn Test

The number of left turns in the corner turn test significantly correlated with the brain water content of the right-frontal lobe 24 h following SBI (ρ = −0.70, p < 0.05, Fig. 2c).

Beam Balance Test

The distance traveled for the beam balance test also significantly correlated with brain water content (ρ = −0.35, p < 0.05, Fig. 2d).

Discussion

SBI pathophysiology develops over several hours after the initial insult. The primary injury consists of the initial lobectomy and intraoperative hemorrhage [11]. Secondary injury occurs within hours of resection and includes brain edema, neuroinflammation, and postoperative hematoma [10, 11]. As with most neurological diseases, brain edema is a predictor of poor outcome in patients. Therefore, development of novel therapies for reducing or preventing cerebral edema is key to lowering the costs of SBI.

The rat model of SBI, developed by Vikram et al. [11], mimics the damage to healthy tissue that is unavoidable during many neurosurgical procedures. Post SBI, rats experience an increase of 4–5 % in brain water content of the right-frontal lobe, causing tissue swelling [11]. The current study utilized the rat SBI model to identify the appropriate sensorimotor tests for evaluating drug efficacy after SBI.

Herein, four common and inexpensive sensorimotor tests were examined for their ability to predict the magnitude of cerebral edema after SBI. The composite Garcia neuroscore is a combination of several subtests evaluating sensorimotor function [5, 13]. Furthermore, the composite Garcia neuroscore has the benefit of identifying deficits caused by unilateral injury because a number of the tests examine left versus right function, such as axial sensation and limb movement symmetry [5, 13]. Despite the fact that statistically significant functional deficits are found between SBI-injured animals and sham animals (Fig. 1b), they did not correlate with the brain water content. This is likely due, in part, to the inclusion of neuroscore subtests that do not identify unilateral injury but rather the presence of an injury, such as spontaneous movement. It is important to note that the vast majority of SBI-injured animals scored perfectly on the spontaneous activity subtest, which has previously been observed for unilateral injuries [13].

The beam walking test examined motor coordination and balance. Although the beam walking test found a difference in the ability of injured animals to cross to a platform compared with sham animals (Fig. 1c), no correlation was observed between beam walking score and brain water content. Despite the lack of correlation for the beam walking test, a trend is apparent, suggesting that an injury is present, but the uninjured side was likely able to compensate for the deficits in an animal’s ability to complete the beam walking test. Our results for the beam walking test are similar to other unilateral injuries, specifically intracerebral hemorrhage, which found that brain water content did not correlate with the function of rodents on the beam walking test [13].

The corner turn test was first used to examine functional deficits after focal cerebral ischemia in mice [22]. The animal’s tendency to turn in a particular direction is measured using the corner turn test, and after a unilateral injury, such as SBI, animals are expected to turn toward the ipsilateral side due to functional deficits in the contralateral limbs [19]. The nature of the SBI rodent model argues for use of the corner turn test, and is supported by the significant correlation between corner turn functional deficits and brain water content in rats after SBI. The beam balance test, which examines proprioception and balance deficits, has many variations, increasing sensitivity for finding marked differences in the performance of injured versus noninjured animals. Herein, we utilized the standard horizontal plane and used the distance traveled as the measure of injury. Although the beam balance test is not inherently a test used to identify unilateral injury, we observed that the functional deficits correlated with brain water content. Similar to the beam walking test, animals may be able to compensate for unilateral deficits, preferring to use the uninjured side. However, the amount of time allotted for testing with the beam balance test (3 min) may have increased sensitivity, making the neurobehavioral deficits apparent in the analysis of distance traveled during this task.

In conclusion, the brain water content of SBI-injured rats was found to significantly correlate to the functional deficits in the corner turn and beam balance tests. No correlation was found for the neuroscore and beam walking score and brain water content. This study suggests that the most appropriate neurofunctional tests for post-SBI behavioral analysis are the corner turn and beam balance tests. Further studies include examining additional cost-effective neurobehavior tests for correlation between functional deficits and brain edema.