Midazolam Intratecal

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European Journal of Anaesthesiology 2005; 22: 907912 2005 European Society of Anaesthesiology

ISSN 0265-0215

Original Article

Neurotoxicity with single dose intrathecal midazolam

administration

B. Ugur*, K. Basaloglu, T. Yurtseven, U. Ates, O. N. Aydin*, D. zen, M. Yurtseven, A. Grel*

Adnan Menderes University Medical Faculty, Departments of*Anaesthesiology and Reanimation, Histology andEmbryology, Aydin; Ege University Medical Faculty, Departments ofNeurosurgery, Histology and Embryology,Izmir, Turkey

SummaryBackground and objective: The aim of study was to investigate the electron microscopic changes in the medulla ofthe spinal cord that occur with intrathecal midazolam administration. Methods: Twenty-eight albino rabbits ofNew Zealand type were randomized into two groups. Following anaesthesia, 16 rabbits were given 300g of mida-zolam (Group M) and 12 rabbits were given 0.3 mL of normal saline solution (Group C) intrathecally. Eightrabbits from Group M (Group M1) and 6 rabbits from Group C (Group C1) were sacrificed 24h after the anaes-thesia and 8 rabbits from Group M (Group M2) and 6 rabbits from Group C (Group C2) were sacrificed 6 daysafter the anaesthesia. The lumbosacral portion was removed by laminectomy and thin sections were examinedmicroscopically. Results: Severe separation in myelin lamella of the large axons, honeycomb appearance, slightseparation in myelin lamella of small to moderately large axons, degenerate vacuoles in the cytoplasm andnuclear membrane irregularity were observed in neurons of Groups M1 and M2. Myelin lamella and nuclearmembranes were found to be regular, vacuoles and oedema were observed in the neurons in the Groups C1 andC2. Conclusion: Midazolam administered at single dose by the intrathecal route may have neurotoxic effects on

the neurons and myelinated axons at 24 h and 6 days following administration.

Keywords: MICROSCOPY ELECTRON; MIDAZOLAM; INJECTIONS SPINAL, intrathecal; NEUROTOXICITY;RABBITS.

Introduction

Intrathecal and epidural drug administration for post-operative analgesia and relief of cancer pain is widely

used. Various drugs have been administered and eval-uated for intrathecal or epidural routes in animals andhuman beings [13]. The first drugs were opioidswhich were followed by -2 agonists such as cloni-dine, local anaesthetics and several peptides [46].The development of spinal drugs such as midazolam

that optimize antinociceptive effects and minimizeadverse effects therefore seems desirable as an alter-native to opioids or local anaesthetics in morphine-tolerant patients [7]. Midazolam is a widely used

systemic adjuvant delivered for its sedative, anxiolyticand amnesic effects [8,9].

Benzodiazepines may modulate the affinity of-aminobutyric acid (GABA) at its receptors whileenhancing its control of chloride channel activity [10].When intrathecally administered, they have relievedpain of somatic origin, produced selective sensoryblockade and blocked somatosympathetic reflexes [7].It is important to perform neurotoxicity screening inanimals of compounds intended for spinal adminis-tration before performing clinical experiments in

Correspondence to: Bakiye Ugur, Adnan Menderes Universitesi, Tip Fakltesi,Anesteziyoloji ve Reanimasyon Departmani 09100, Aydin, Turkey. E-mail:[email protected]; Tel:90 256 444 1256; Fax:90 256 214 6495

Accepted for publication September 2005 EJA 3058


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human beings. However, there is still controversyregarding its possible neurotoxicity to the spinalcord.

Some animal studies have shown neurotoxicity ofspinally [2,11] and epidurally [12] administered mida-zolam in rabbits; but some others not [13,14].

Possible toxic reactions such as demyelination,axonal degeneration, oedema, necrosis, arachnoiditis,

vascular changes [15], vacuolization and fibrosis [12]may be related to the drug itself or to chronic admin-istration of drugs via long-term cannulation duringthe study. Thus, it is difficult to distinguish thedamage induced by midazolam from that induced bythe catheter. Furthermore, it is not clear whether thisneurotoxicity is due to the drug itself, preservativehypoxia or ischaemia [4]. In addition, the efficacy oflight microscopy in determining neurotoxicity of adrug is a matter of debate [16]. There are few studiesinvestigating the histological changes by electronmicroscopy.

The purpose of the present study was to investi-gate the histological reactions of the spinal cord at24 h and 6 days to directly administered single dosemidazolam in rabbits by electron microscopy.

Methods

Following approval of the animal Ethics Committee,28 New Zealand albino rabbits of either sex, weigh-ing 2.32.5kg were randomly assigned into twogroups: Group midazolam (M) received 300 g(0.3 mL) midazolam (Dormicum; Roche, pH 3.3,1mgmL1, n 18), and group control (C) received0.9% saline (pH 5.5, 0.3mL, n 12) intrathecallyfollowing laminectomy under anaesthesia.

The rabbits were anaesthetized with 5mg kg1

xylazine (Alfazyne 20 mgmL1; Alfasan InternationalB.V.) and 10mgkg1 ketamine (Ketalar; Parke-Davis, 50mgmL1) by ear veins. All rabbits were keptin identical conditions with respect to food regimen,temperature, humidity and periods of daylight.

A pulse oximeter probe was placed on the rabbitsear after shaving the area and heart rate (HR) andperipheral oxygen saturation (SPO2) were monitored.The exclusion criteria were an HR of lower than

130min1 and an SPO2 below 90.Eight randomly selected rabbits from the mida-

zolam group (Group M1) and six rabbits from thecontrol group (Group C1) were sacrificed after 24 h.The remaining eight rabbits from the midazolamgroup (Group M2) and six rabbits from the controlgroup (Group C2) were sacrificed after 6 days byadministration of transcardiac 1% formaldehyde solu-tion and 2% gluteraldehyde with 0.1mol phosphatebuffer. The spinal medulla of the rabbits were removedby laminectomy.

Surgical procedure

The lumbosacral areas and surgical sites of the rab-bits were incised on the midline following steriliza-tion with 10% iodine solution. The laminectomyprocedure was carried out with the aid of an operat-ing microscope through L7S1 intervertebral spacefollowing exploration of the lumbosacral junction.The dura was fixed and the injection was performedintrathecally through a 22-G needle with an insulinsyringe. Muscle, fascia and skin layers were closedand sterilized again with iodine solution.

Lumbosacral specimens removed from the site ofintrathecal administration and exposed to post-fixationwith 1% osmium tetroxide following fixation with2.5% gluteraldehyde. They were embedded intoepon (812) following routine electron microscopicprocedures. Thin sections of epon blocs obtainedfrom a Reicter ultra microtome were stained byuranyl acetate and lead citrate. Specimens wereexamined by a histologist who was blinded to the

injected solution and the time of sacrifice.All rabbits were observed for sleeping habits, food

intake and motor activity. The motor activity wasassessed by observing the rabbits moving freely intheir cages and the reaction provoked by suddenmovement or the clapping of hands.

Results

No behavioural changes were observed in the animalsfollowing midazolam or normal saline administrations.In none of the rabbits SPO2 values reduced below 90and HR reduced below 130min1 during the admin-istration. SPO2 varied from 95 to 100.

The electron microscopic evaluations of Group M1(n 8) revealed severe separation and degenerationin myelin rings of large myelinated axons, vacuoleformation and oedema in the cytoplasm (Fig. 1), ahoneycomb appearance in the myelin lamella, dis-organization, slight separation in myelin lamella ofmoderately large and small axons, vacuole formationand oedema in the cytoplasm (Fig. 2), irregularitiesand invaginations of nuclear membrane (Fig. 3) at24 h in all preparations.

In Group C1 (n 6) specimens, myelin lamellaand nuclear membranes were regular and a few smallvacuoles were observed in the neurons. A specimenfrom a rabbit sacrificed at 24h after saline injection isshown in Figure 4.

In Group M2 (n 8) specimens, severe separationand degeneration in myelin lamella of large axons,vacuoles and oedema in the cytoplasm (Fig. 5),honeycomb view in myelin lamella of large axons,slight separations and disorganizations in the mod-erately large axons, very slight degeneration in the

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myelin lamella of small axons (Fig. 6), irregularity andinvaginations of nuclear membrane and a few, moder-ately large degenerated vacuoles and oedema (Fig. 7)were observed in the grids taken on the 6th day.

In Group C2 (n 6) specimens, myelin lamella ofaxons and nuclear membranes were regular (Fig. 8),and vacuoles and oedema in the cytoplasm (Fig. 9)were observed in the neurons. Vacuole formation andoedema in the cytoplasm was the common histolog-ical changes observed in all groups.

Discussion

The lumbar spinal canal is a region which allowsinjection of various solutions including the anaes-thetic agents and of which subarachnoid and epiduralspaces allow catheter insertion. Various studies have

Neurotoxicity with midazolam 909


Figure 1.In the Group M1 separation of myelin rings ( ) vacuoles andoedema ( ).

Figure 2.In the Group M1 honeycomb view of myelin lamella, slight separa-tion in myelin lamella of small axon, vacuole and oedema in thecytoplasm.

Figure 3.In the Group M1 nuclear membrane irregularities, invaginations( ).

Figure 4.In the Group C1 regular nuclear membrane ( ), regular myelinlamella ( ), vacuoles and oedema in the cytoplasm ( ) (7200).


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shown that analgesia might be provided by intrathe-cal or epidural administration of midazolam in humanbeings in the postoperative period and chronic backpain [3,1720].

Studies on neurotoxic effects of intraspinally admin-istered drugs are of utmost importance. Interestingly,

these drugs are generally tested for clinical side-effectsprior to their administration, but not for neurotoxiceffects. There are few studies on the neurotoxicity ofintrathecally administrated midazolam in animals.

Although animal toxicity studies have yielded con-flicting results, the present study is consistent withthose studies that found that intrathecal midazolamdid cause neurotoxicity [2,11,12] and were differentfrom those that suggested it did not [13,14,21].

Our results have shown that intrathecal singledose midazolam without chronic catheter adminis-tration caused clear histological changes in specimensobtained from spinal medulla by laminectomy after24 h and 6 days following administration.

We found separation in myelin lamella of small,moderate and large axons and honeycomb appear-ance, disorganization, nuclear membrane irregular-ity and vacuole formation in all of the sections afteradministrating midazolam. Myelin and nuclearmembranes were regular but there were vacuoles in

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Figure 5.In the Group M2 severe separation in myelin lamella ( ), vac-uoles and oedema in the cytoplasm ( ) (7200).

Figure 6.In the Group M2 honeycomb view in myelin lamella ( ), slightseparations in the small to moderately large axons ( ) (7200).

Figure 7.In the Group M2 nuclear membrane irregularity and invaginations( ), vacuoles and oedema ( ) (5400).

Figure 8.

In the Group C2 regular nuclear membrane and myelin lamella.


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the cytoplasm in the sections from the saline groups.Vacuole formation and oedema in the cytoplasmwere the common findings in both saline and mida-zolam groups. We believe that these changes mightbe attributable to the midazolam administration.

Malinovsky and colleagues [2] reported necrosis,haemorrhage and other histopathological changes intwo of nine spinal cords of rabbits that had received asingle intrathecal injection of midazolam of 300g(0.3mL) by the intracisternal route by light and fluo-rescence microscopy. Although it is impossible toperform blind percutaneous puncture in the rabbitlumbar region without causing trauma, this is easier inthe larger cervical subarachnoid space. Therefore, theauthors preferred the intracisternal route.

In contrast, Nishiyama and colleagues [14] reportedno inflammatory reaction of the spinal cord in cats bylight microscopy, in the acute phase up to 6 h afterthe direct administration of high doses of midazolamof 10mg. They suggested that 6 h might not be longenough to see acute histological changes that wouldresult in chronic changes. Also by light microscopy,Serrao and colleagues [13] did not find damage to thespinal cord, nerve roots or meninges and evidence oftoxicity by administering to the rats midazolam for

48 weeks via an indwelling intrathecal catheter.Although there was no statistically significant differ-ence when midazolam was compared with saline, theyobserved pathological changes to some extent in bothgroups and they attributed this finding to the mechan-ical presence of the catheter as in studies by Johansenand colleagues [21] in their sheep model by lightmicroscopy. Indeed, chronic spinal cannulation hasbeen shown to lead to changes, including fibrosisand lymphocytic infiltrations around the catheter inthe subarachnoid space [4]. We administered drugs

intrathecally by a laminectomy method undermicroscopic control in order to minimize thetrauma.

Bozkurt and colleagues [12] administered a singledose of acidic midazolam (commercially availablepreparation) epidurally in neonatal rabbits. In thiselectron microscopic study, an acidic saline vehiclecontrol (matching the pH of the midazolam) was

used, and pathology was observed with the acidicvehicle formulations that exceeded the pathologyobserved with a non-acidified vehicle. They pointedout that one of the concerns when applying mida-zolam to the spinal cord in animal trials was thespecies and age differences of animals because thedeveloping spinal cord where myelination is notcomplete might be more sensitive to midazolam.

Johansen and colleagues [21] demonstrated no spinalcord toxicity after 43 days of daily 515mg doses ofintrathecally administered midazolam in a sheepmodel by light microscopy. The single histological

finding in this study was characterized as a mild tomoderate infiltration of inflammatory cells surround-ing the catheter tract; this was present in all animals.They suggested that this inflammation was likelyattributable to the mechanical presence of the catheter.

In contrast to Johansen and colleagues [21] work,Erdine and colleagues [11] concluded that chronicintrathecal administration of midazolam should beavoided in human beings. They also suggested thatthe histological and vascular lesions in the spinalcord were well correlated with the use of midazolamand preservative free midazolam.

In conclusion, we should consider that whileintrathecal midazolam in human beings might pro-vide positive effects on the analgesia, it may producehistological changes in the spinal cord. In our opin-ion, midazolam may cause neurotoxic effects onmyelinated axons and nuclear membranes, but wealso believe that further studies are required fordetermining whether these changes could cause irre-versible neurological damage.

Acknowledgements

This study was funded by Research Fund Accountancyof Adnan Menderes University. This study was pre-sented orally at XXXVII. Turkish Anaesthesiologyand Reanimation Society National and II. Interna-tional Congress.

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Figure 9.In the Group C2 regular nuclear membrane, vacuoles in the cyto-plasm, regular myelin lamella.


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