Neurol Sci (2012) 33:1155–1160 DOI 10.1007/s10072-012-0930-3

ORIGINAL ARTICLE

The changes of brainstem auditory evoked potentials (BAEP) after vertebrobasilar artery ischemia in rabbits Zeng-Lin Cai • Zheng-Chun Zhang • Jian-Qiang Ni • Shou-Ru Xue • Li-Zhen Xu Fang-Ping Wu

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Received: 4 February 2010 / Accepted: 2 January 2012 / Published online: 31 January 2012 Ó Springer-Verlag 2012

Abstract Brainstem auditory evoked potentials (BAEPs) have been used as a valuable neurophysiologic index of neuronal dysfunction in the level of the brainstem. BAEPs are also useful in subdividing evoked potentials into normal, slight, or pronounced in patients with vertebrobasilar insufficiency. We investigated the changes of BAEP after vertebrobasilar artery ischemia in rabbits and its significance in clinical work. A brainstem ischemic model was made by unilateral extracranial occlusion of vertebral artery to monitor BAEPs at 0, 10, 20, 30, 40, 50, and 60 min after occlusion. We found that peak latencies (PL) of I, III, and most notably V were gradually extended. In addition, we observed a significant (P \ 0.05) delay of interpeak latencies (IPL) of waves I–III, III–V, and I–V after occlusion. This delay became more significant in IPL I–V 60 min after occlusion. Our results also demonstrate that the amplitude of I and V had no obvious change (P \ 0.05). In the rabbit with bilateral extracranial occlusion of vertebral artery, BAEP waveforms disappeared 10 min after occlusion. Our results showed that vertebrobasilar insufficiency caused brainstem ischemia, which induced BAEP abnormity. Taken together, our findings suggest that BAEP has important significance for the clinical diagnosis of vertebrobasilar insufficiency. Therefore, early detection of neuronal change after transient

Z.-L. Cai
F.-P. Wu Department of Neurology, Affiliated Lianyungang Hospital of Xuzhou Medical College, Lianyungang 222002, China e-mail: [email protected] Z.-L. Cai
Z.-C. Zhang (&)
J.-Q. Ni
S.-R. Xue
L.-Z. Xu Department of Neurology, First Affiliated Hospital of Soochow University, Suzhou 215004, China e-mail: [email protected]

cerebral ischemia is important in initiating treatment within the therapeutic window. Keywords Brainstem auditory evoked potentials (BAEP)
Vertebrobasilar insufficiency (VI)
Peak latencies (PL)
Interpeak latencies (IPL)

Introduction Brainstem auditory evoked potential (BAEP) has been used as a valuable neurophysiologic index of neuronal dysfunction in neurology, neurologic surgery and otology, since first described by Jewett and Romano [1] and Markand [2]. BAEP is a remarkably stable and robust potential, and is largely resistant to the level of consciousness, sedative medications, and general anesthesia. In addition, BAEP is useful as an intraoperative monitoring tool during cerebellopontine angle tumor surgery [3–5], as a prognosticator of coma [6, 7], and in assisting the diagnosis of brain death [8]. BAEP is a non-invasive and easily reproducible method sensitive to subclinical alterations. A number of studies have emphasized BAEPs importance in the diagnosis of vertebrobasilar failure, offering an additional tool to the obligatory morphological investigations including CT and MRI scans. Importantly, the BAEP method is capable of preserving function of different nuclei and pathways, and conversely, can unveil impaired function even when morphological integrity is compromised [9]. Additionally, BAEP has been shown to subdivide evoked potential changes into normal, slight, or pronounced in patients with vertebrobasilar insufficiency [10]. For example, in patients with transient ischemic attack (TIA), transient abnormal BAEP was observed during resolution of ischemia, which can further identify the

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suggested continuum between ischemia and infarction in the vertebrobasilar circulation [11]. In this study, we investigated the effects of vertebrobasilar insufficiency on BAEP in a rabbit model. We ligatured the unilateral vertebral artery, reducing the blood supply of the basilar artery, causing brainstem ischemia, while continually recording the change of BAEP up to 1 h after ligaturation. Our findings suggest that BAEP has important clinical significance in diagnosing vertebrobasilar insufficiency, and BAEP is useful as a monitoring tool during acute basilar artery infarct.

Materials and methods Animal preparation This study was approved by the Soochow University Ethics Committee. 16 overnight-fasted New Zealand White rabbits weighing between 2.3 and 3.2 kg were used. Food and water were available ad libitum. Animals were anesthetized with 25% urethane (1 g/kg), and tilted at *30° to the horizontal to facilitate access to the vertebral arteries. A midline posterior cervical incision and approach were made, and the posterior elements were exposed out to the lateral edge of the lateral masses, and then the vertebral arteries were exposed just before their entry into the transverse foramen of the cervical vertebra. A tracheostomy was performed in the anterior triangle of the neck, and the animals were placed on a respirator. The animals were kept normoxic and normocarbic during the surgical procedure. Experimental group

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animals were killed at the same time. Blood loss for the surgical preparation did not exceed 5 mL. Recording of brainstem auditory evoked potential (BAEP) All BAEP data were recorded with an auditory evoked potential system (Nicolet, Viking IV, Madison, WI, USA). Briefly, needle electrodes and reference electrodes were placed at A1 and A2 with Fpz as ground electrode. Recording electrodes were placed at Cz. Alternating polarity clicks of 0.1 ms duration were presented monaurally in a rhythm of 11.4 stimulations; polarity rarefied in the intensity of 100 dB normal hearing level (NHL) in both ears separately. Each response was amplified (9100,000), filtered at 100–3,000 Hz and stacked in runs of 1,000 sweeps on average. Two registrations of each ear were carried through to confirm reproducibility of the waves and tracing overlapping. The impedance values of the electrodes were confirmed to be below 5 kX. The BAEPs were recorded using a computer-based electrodiagnostic system and were stored on computer disk. Latency was measured from the stimulus onset and wave form amplitude, which was measured from one peak to the peak of opposite polarity immediately following it, using values derived from BAEP recordings from each ear of cattle in both groups. Pathological section observation The post-fixed brains were protected in 25% sucrose in PBS. Brains were coronally sectioned on a cryostat at 20 lm thickness or paraffin-embedded and sliced on a microtome at 6 lm thickness. For histological assessment of damage, the paraffin-embedded brain sections were stained with HE (hematoxylin–eosin staining).

After 12 healthy New Zealand rabbits were fixed and sheared, a longitudinal endlong incision was made above the first costal margin, and connective tissue was peeled off. Bilateral neck region muscle were ligated and cut off, and nervus and vascularis were separated, and then brachial plexus were located. Subclavian arteries were carefully separated from brachial plexus. The right-side vertebral artery was clipped with artery clamp, and BAEPs were recorded at 0, 10, 20, 30, 40, 50, 60 min after occlusion. All rabbits were killed by injecting potassium chloride 6 h after induction of ischemia. The medulla oblongata of rabbits were removed, trimmed by transverse sectioning with a razor blade (ca. 5 mm rostral to 5 mm caudal from the obex), and then put into 20% formaldehyde solution.

Results are expressed as mean ± SEM. Statistical significance between groups was assessed by ANOVA followed by Dunnett’s t tests. Significance of P was defined as value \0.05.

Control group

Alterations of waveform

4 healthy New Zealand rabbits were treated as described above, but vertebral arteries were not ligatured. Sham

Experimental group In rabbits with bilaterally evoked BAEPs, the absolute latencies of waves I, II, III, IV, and V

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Statistical analysis

Results Alterations in brainstem auditory evoked potentials

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were delayed, as were parts of waves III and V were poorly differentiated and had poor repeatability (Table 1; Fig. 1a–c). Control group BAEPs in both sides of all rabbits were recorded. PL and interpeak latencies (IPL) amplitude values at all time points had no statistical significance (P [ 0.05) compared with preoperative BAEP. Alterations of peak latencies (PL) Compared with the control group, we found significant prolongations for PL I, III, and V, respectively (P \ 0.05), especially PLV (Table 1; Fig. 2a). PL I The latencies were significantly increased at 30 (P = 0.042), 40 (P = 0.025), 50 (P = 0.048), and 60 min (P = 0.018) after ischemia compared with preoperative values. The minimum value prolonged was 0 ms 60 min after ischemia, and the maximum was 0.60 ms. The alterations in the right side were significant compared with the left (P = 0.001). PL III The latencies were significantly increased at 30 (P = 0.001), 40 (P = 0.017), 50 (P = 0.011), and 60 min (P \ 0.001) after ischemia compared with preoperative values. Post-ischemia 60 min, the minimum value prolonged was 0.19 ms, and the maximum was 2.00 ms. The changes in the right side were significant compared with the left (P = 0.037). PL V The latencies were significantly increased at 20 (P = 0.050), 30 (P = 0.002), 40 (P \ 0.001), 50 (P \ 0.001), and 60 min (P \ 0.001) after ischemia compared

with preoperative values. The minimum value prolonged was 0.45 ms, and the maximum was 3.85 ms 60 min after ischemia. The changes in both sides had no significant difference (P = 0.967). Alterations of interpeak latencies (IPL) IPL I–III, III–V, and I–V were significantly increased compared with the control group (P \ 0.05), especially IPL I–V (Table 1; Fig. 2b). IPL I–III The latencies were significantly increased at 30 (P = 0.013), 40 (P = 0.012), 50 (P = 0.018), and 60 min (P = 0.017) after ischemia compared with preoperative values. The minimum value prolonged was 0 ms, and the maximum value was 1.46 ms 60 min after ischemia. The changes of both sides had no significant difference (P = 0.866). IPL III–V The latencies were significantly increased at 40 (P = 0.019), 50 (P = 0.005), and 60 min (P = 0.005) after ischemia compared with preoperative values. The minimum value prolonged was 0.13 ms, and the maximum value was 3.17 ms 60 min after ischemia. The changes of both sides had no significant difference (P = 0.202). IPL I–V The latencies were significantly increased at 30 (P = 0.025), 40 (P = 0.001), 50 (P = 0.001), and 60 min (P \ 0.001) after ischemia compared with preoperative values. 60 min after ischemia, the minimum value prolonged was 0.17 ms, and the maximum value was 4.12 ms. The changes of both sides had no significant difference (P = 0.290).

Table 1 BAEP changes of rabbits after right-side vertebral artery ligatured

PL I (ms) PL III (ms)

Sides

0 min

10 min

20 min

30 min

40 min

50 min

60 min

P 0.018

Left

1.34 ± 0.15

1.44 ± 0.15

1.45 ± 0.16

1.48 ± 0.15

1.50 ± 0.16

1.47 ± 0.16

1.49 ± 0.14

Right

1.43 ± 0.17

1.51 ± 0.19

1.57 ± 0.24

1.61 ± 0.28

1.63 ± 0.29

1.64 ± 0.28

1.69 ± 0.28

Left

2.96 ± 0.21

3.14 ± 0.27

3.19 ± 0.30

3.31 ± 0.42

3.33 ± 0.42

3.32 ± 0.37

3.37 ± 0.41

Right

2.98 ± 0.23

3.20 ± 0.27

3.29 ± 0.24

3.49 ± 0.48

3.54 ± 0.49

3.47 ± 0.33

3.61 ± 0.51

PL V (ms)

Left

4.28 ± 0.35

4.60 ± 0.43

4.74 ± 0.39

5.02 ± 0.58

5.21 ± 0.61

5.38 ± 0.76

5.86 ± 1.30

Right

4.26 ± 0.33

4.59 ± 0.42

4.80 ± 0.53

5.03 ± 0.84

5.16 ± 0.69

5.43 ± 0.73

5.85 ± 0.84

IPL I–III (ms)

Left

1.65 ± 0.17

1.71 ± 0.19

1.76 ± 0.21

1.84 ± 0.29

1.82 ± 0.32

1.85 ± 0.24

1.89 ± 0.30

Right

1.56 ± 0.20

1.71 ± 0.24

1.73 ± 0.20

1.89 ± 0.36

1.92 ± 0.38

1.84 ± 0.30

1.91 ± 0.42

IPL III–V (ms) IPL I–V (ms) AMP V (lV)

0.002 0.000 0.017

Left

1.27 ± 0.18

1.44 ± 0.20

1.55 ± 0.25

1.71 ± 0.34

1.89 ± 0.40

2.07 ± 0.75

2.49 ± 1.21

Right

1.33 ± 0.23

1.40 ± 0.20

1.52 ± 0.34

1.54 ± 0.39

1.62 ± 0.29

1.96 ± 0.64

2.26 ± 0.58

0.000

Left Right

2.92 ± 0.31 2.91 ± 0.33

3.16 ± 0.36 3.10 ± 0.36

3.31 ± 0.35 3.25 ± 0.46

3.54 ± 0.49 3.42 ± 0.71

3.70 ± 0.57 3.54 ± 0.59

3.91 ± 0.76 3.80 ± 0.81

4.49 ± 1.45 4.17 ± 0.85

0.000

Left

0.49 ± 0.29

0.61 ± 0.36

0.65 ± 0.26

0.46 ± 0.24

0.79 ± 0.71

0.57 ± 0.39

0.53 ± 0.34

0.919

Right

0.53 ± 0.25

0.46 ± 0.37

0.54 ± 0.34

0.61 ± 0.48

0.54 ± 0.34

0.54 ± 0.21

0.51 ± 0.27

Peak latencies (PL) of I, III and V delayed, especially PL V. Significant (P \ 0.05) delay of interpeak latencies (IPL) of waves I–III, III–V and I– V was observed after occlusion. This delay became more significant in I–V 60 min after occlusion. The amplitudes of I and V showed no significant changes (P \ 0.05)

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Fig. 1 BAEP monitoring. a Continuous BAEP monitoring of a rabbit: peak latencies of I, III, and V gradually delayed. b BAEP of a rabbit during operation until death. A rabbit’s right-side vertebral artery was injured at 0 min and then the vertebral arteries of both sides were ligatured at 10 min; the rabbit died after 10 min. All waves were gradually prolonged until disappearance at death. c Changes in wave V

Alterations of amplitude (AMP) At all time points of experimental group and control group, alterations of all wave’s amplitude were not statistically significant (all P [ 0.05). Alterations of pathology In neurons in the bulbus medullae in experimental groups, we observed cell swelling, vacuolization at cell periphery, disappearance of some tigroid bodies, neuronophagia phenomenon, and aggregation of chromatin into dense staining. While in control group such changes were not observed (Fig. 3).

Discussion Brainstem auditory evoked potentials are short-latency potentials recorded from the surface of the head during a brief acoustic stimulation. These potentials, consisting of five vertexes recorded positive peaks (waves I–V) within 10 ms of the stimulus onset, are routinely used in clinical practice to evaluate the normality of the lower auditory system [12]. Because the auditory pathway is composed of crossed and uncrossed fibers within the brainstem, multiple

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interconnections exist at all levels. It is still not entirely clear to what extent BAEP components are generated within the respective ipsilateral and contralateral pathways. However, it is certain that waves I and II are generated ipsilaterally to the stimulated ear in regions before any decussation occurs [13]. Spiral ganglion cells of the cochlea generate wave I, while wave II is generated by the cochlear nucleus cells. The latencies of waves III, IV, and V in the rodent BAEP seem to be consistent with activity recorded from the cochlear nucleus and contralateral superior olivary complex (SOC) cells, ipsi and contralateral cells of the SOC, the lateral lemniscus, and/or the inferior colliculus, respectively [12, 14]. BAEP recordings are clinically useful and are noninvasive procedures, given that they can represent a stress to the early impairment of the auditory nerve or brainstem. Therefore, BAEP has been widely used to objectively evaluate the lower auditory system disease, to diagnose acoustic neurinomas, and lesions of the brainstem, such as multiple sclerosis, vertebrobasilar artery ischemia, and infarct [12, 15]. Our data indicate several important points: (1) after right-side vertebral artery was ligatured, all waves of BAEP were gradually prolonged, and the PL I, PL IIIs changes in the right side were significant compared with the left. We argue these results are due to the interrupted

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Fig. 2 Measured changes in BAEP latencies. a Peak latency changes of experimental group. Significant prolongations were seen for PL I, III, and V, respectively (P \ 0.05), especially PL V. The changes of PL I and PL III in the right side were significant compared with the left (P = 0.001 and 0.037). b Interpeak latency changes of experimental group. IPL I–III, IPL III–V, and IPL I–V were significantly increased compared with the control group (P \ 0.05), especially IPL I–V. The changes of both sides had no significant difference

blood flow. Inner ear blood is supplied by internal auditory artery, which vertically comes from anterior inferior cerebellar artery or basilar artery, then emanates branches reaching the root of the auditory nerve [16]. Because it is slender and lacks collateral circulation, the internal auditory artery is very sensitive to ischemia. (2) The prolongation of PL V appeared earliest at 20 min, whereas PL I

Fig. 3 Representative photomicrographs of HE staining (a–c). a Normal bulbus medullae brain tissue. b Bulbus medullae tissue appeared with cell swelling, vacuolization in cell periphery, some

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and PL III were not observed until 30 min post-ischemia. Again, the functional flow of blood is a factor of cause. The blood supply of pons cerebelli is provided by pontine arteries with three small branches including median artery, short circumflex artery, and long circumflex artery. According to the fluid mechanics principle, the smaller the ratio (branch caliber:trunk caliber), the larger the branch angle, resulting in higher fluid resistance. Thus, when vertebrobasilar artery blood supply is insufficient, it causes auditory pathway ischemia, resulting in our observed PL V prolongation earlier than other wave’s latency, and its poorly differentiated waveform. (3) Changes of IPL had statistical significance; among these IPL I–V prolonged most remarkably. When ischemia and anoxaemia change the membrane potential, the ability of nerve cells to generate and conduct electrical impulses degrades. Therefore, every interpeak latency of BAEP showed delays. (4) In our experimental group, the features of the neurons in the bulbus medullae appeared to show cell swelling, vacuolization in cell periphery, some tigroid body disappearance, neuronophagia phenomenon, and aggregation of chromatin into dense staining. The above data indicate that vertebrobasilar insufficiency can cause brainstem ischemia, inducing BAEP abnormity. These data underscore BAEPs important clinical significance in the diagnosis of vertebrobasilar insufficiency and the functional restoration of brainstem after vertebrobasilar infarct. Early detection of irreversible neuronal change after transient cerebral ischemia is critical in adequately treating patients within the therapeutic window. Discrepancies between morphological and functional images are commonly observed when evaluating stroke and pre-stroke cases. Our observation that brainstem infarction affected the auditory pathway that resulted in both BAEP positive and BAEP negative findings, is in line with expectations [17].

tigroid body disappearance, and aggregation of chromatin into dense staining. c two phenomenons of neuronophagia (right arrow)

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For example, Muttaqin et al. [18] used a brainstem ischemic model by injecting cylindrical silicone embolus to the right vertebral artery to monitor the brainstem auditory evoked responses (BAERs). In embolized animals, significant delay of IPL of waves I–III, III–V, and I–V was observed 15 min after embolization. This delay became more significant 30 min after embolization. They considered BAER monitoring could be used to complement other diagnostic methods for patients with vertebrobasilar insufficiency or infarction. In addition, Drake et al. [19] recorded BAEPs in 35 patients with TIAs in the vertebrobasilar system that did not have a stroke. Wave V was significantly longer in latency and lower in amplitude in TIA patients. They noticed occasional BAEP abnormality during the resolving transient ischemia and they suggested continuous monitoring between ischemia and infarction in the vertebrobasilar territory. Finally, Rao and Libman [20] described a patient with isolated vertigo with abnormal BAEPs. The patient subsequently developed an anterior inferior cerebellar artery territory infarct. They suggested that BAEP testing might lead to early recognition in those patients at risk for catastrophic stroke, and prompt appropriate investigation and treatment to prevent this outcome. In summary, we made a vertebrobasilar insufficiency model by unilateral extracranial occlusion of the vertebral artery and monitored the BAEP. Our results show that vertebrobasilar insufficiency can cause brainstem ischemia, which induces abnormal BAEPs. We argue that BAEP has important significance for the clinical diagnosis of vertebrobasilar insufficiency and the functional restoration of brainstem after vertebrobasilar infarct. In order to provide adequate treatment within the therapeutic window, early detection of irreversible neuronal change after transient cerebral ischemia is important. Acknowledgments We thank Neural Electrophysiology Department of First Affiliated Hospital of Soochow University for technical support in the experiments. We also want to thank Dr. Austin Cape for careful reading and insightful suggestions.

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