Histological Changes of the Sciatic Nerve in Dogs After Intraneural Application of Lidocaine – Relation to the Established Application Pressure

Histological changes of sciatic nerve in adult dogs  days after single application of  lidocaine ( ml dose, speed of injection  ml/min) and measurement of the application pressure was studied, with a goal to investigate structural changes of the nerve in relation to the established pressure values. Th e application pressure was determined by using Bio Bench software. In intrafascicular puncture an average application pressure of . ±  kPa was found, and in interfascicular puncture its average value was . ± . kPa, with a note that individual diff erences are regularly present. Seven days after the injection, a nerve dissection was performed and serial sections covering the region of injection’s puncture and bordering proximal and distal zones, in the total length of  cm, were prepared. Th e found changed show the presence of nerves’ fi bers lesions with a strong reactivity of Schwann’s cell, as well as the change of interstitial structure concerning hypercellularity and occurrence of cellular extravasation. Th e covering system of the nerve in the zone of epineurium manifests changes of infl ammatory process and in perineurium a decomposition of lamella layers and the alteration of their tinctorial properties were noticed. A comparison of the found nerve reactivities in intraand interfascicular application showed their one-way alteration, although the lesions were more noticeable in the conditions of intrafascicular application. The damages were mostly expressed in the zone of local application of anesthetic, than distally from it, while the damage to the structure in the proximal part is of the smallest degree.


Introduction
Peripheral nerves are supplied with blood through an internal network of endoneurium blood vessels and an external network of epineurium blood vessels.Th ese two networks anastomose via transperineureal blood vessels.An intact blood-nerve barrier is important for the preservation of internal nerve milieu.Accumulation of the liquid within endoneural space in diff erent pathological conditions results in an increased endoneural pressure, reduction in the blood-nerve fl ow and increase in the permeability for osmotically active macromolecules.Regional anesthesia represents a signifi cant problem from the point of nerve damage, especially the damage to the blood-nerve barrier.Carriers of the negative eff ect are: anesthetic itself, needle's diameter, speed of injection, volume of the injected substance and region of application (, , , , ).A strong pain that patient feels is a sign that the nerve was targeted with the needle during the performance of regional anesthesia.Th at is why it is suggested that the nerve blockage is not performed in anesthetized or deeply sedated patients, because they cannot feel the pain during intraneural application after which nerve damage occurs.High pressure during the application of local anesthetic, as a sign that the needle was introduced intraneurally, can have applicable clinical value which could reduce the risk of neurological injuries ().In spite of their wide use, there are a surprisingly small number of studies dealing with neural toxicity of locally applied anesthetics ().

Material and Methods
For this research, we used  sexually mature male dogs, of mixed breed, with average body mass of  kg and average age - years.Th e dogs were kept in a standardized laboratory conditions.Before the experiment was performed, a premedication of animals with acepromazine (. mg/kg, intravenously), atropine (. mg/kg, subcutaneously) and ketamine ( mg/kg, intramuscularly) was done.In general endotracheal anesthesia (halothane) and aseptic conditions, we surgically approached the nerve between biceps femoris muscle and semitendinosus muscle.We bluntly dissected fascias of those muscles, separated the connections with a retractor and found sciatic nerve.At an angle of  degrees, we intraneurally placed a -gauge needle in the region of nerve, under direct control of optical instrument.By using an automatic infusion pump (PHD ; Harvard Apparatus, Holliston, MA) we applied  ml of  lidocaine (Bosnalijek, Sarajevo) with the speed of  ml per minute.Th e achieved pressure during the application was registered by an in-line manometer (PG ; PSI -Tronics Technologies Inc, Tulare, CA) connected to a computer by an analogue-digital converter (DAQ ; National Instruments, Austin, TX).Th e manometer was placed proximally from the needle with which it was connected by a non-distendible tubes (high durometer polyvinyl chloride injection tubing; '' arterial pressure tubing; Abbot Critical Care System, Abbot Laboratories, North Chicago, IL).Th e data about the pressure were analyzed by using a software package (BioBench version .; National Instruments, Austin, TX).After the application is done, the skin wound was sutured with an "x" stitch of non-absorbable suture.Seven days after the injection, an intravital excision of the nerve from the area of puncture and bordering proximal and distal zones,  cm in length, was performed.Nerve samples were fi xated in  formalin, embedded in paraffi n, sectioned in series and stained with HE method.Prepared histological sections were analyzed under the light microscope with installed digital camera.For the evaluation of the results, standard statistical methods were used.Computer based evaluation of the mean value of pressures is presented as the mean value (X), standard deviation (SD) and standard error of the mean value (SEM).For investigation of signifi cant diff erences between intrafascicular and extrafascicular group we used the t-test.P-value (p<.) was signifi cant.

Results
Calculated mean values of the application pressure in intrafascicular application were (.±  kPa) and in extrafascicular application (. ± . kPa).At the time of lidocaine application in all nerve samples spindle-shape edemas were noticed in the puncture area and in neighboring proximal and distal zones.In the area of application in intrafascicular injections, changes in the histological structure were noticed, including: Perineurium showed division of lamellas with its significant disintegration at the place of puncture and the loss of demarcation toward the surrounding perifascicular connective tissue and closest nerve fi bers.Blood vessels incorporated in its structure and its surroundings are more noticeable but there are no signs of hyperemia and extravasation (Figure ).Nerve fi bers of the fasciculus into which the anesthetic was directly injected showed damages in general, while subperineural ones had more intensive changes of wide range compared to the central ones.Myelin fi bers were disarranged in the space and of increased volume.Some of the axons of these fi bers were dislocated and hyperacidophile.In some of them, an advanced axolysis up to the degree of complete disintegration was noticed.In those cases, residual structureless masses appear in fi ber's structure.In some areas, myelin sheath showed increased acidophilia and, occasionally, loss of normal structure and transformation into a thin, hyperacidophile, structureless ring.Amyelin fi bers were disarranged and fi lled with hyperacidophilic axoplasm ( an increased number of macrophages, lymphocytes, plasma cells, hyperemic blood vessels and bundles of collagen fibers with altered, i.e. uneven tinctorial attributes.Collagen fi bers are more compactly arranged in the perifascicular zone and strongly acidophilic.In the nerve zone which is located distally from the puncture area as well as in the area proximally from the puncture site, histological changes are of the same

Discussion
In rare cases, clinical use of local anesthetics is related to neurological morbidity ().Th e mechanism of the occurrence of neurological sequelae after intraneural application of local anesthetic is not completely clarifi ed.Some authors state that the degree of nerve damage after intraneural application of various agents depends on the type and dose of the agent (, , ).Nerve damages during intrafascicular application of local anesthetic are results of direct trauma during application () or of ischemia which leads to the damage of blood-nerve barrier and endoneural edema ().Th e pressure, as a factor that contributes to the damage, is mentioned, whereby intraneural application is in relation with different levels of application pressure ().
Other authors were mainly dealing with the diff erence in nerve damage during paraneural and intraneural application or with the diff erences in damages during paraneural application of different substances (, ).
We have observed pressure diff erences in intrafascicular and extrafascicular application of local anesthetic, keeping in mind the diff erent structure within the nerve itself.Our data show that the degree of nerve damage is higher in intrafascicular application which is in relation to high application pressure, with mean values found by us were . ±  kPa, while in cases of extrafascicular application those mean values were . ± . kPa, with parallel smaller damages of histological structure.Our previous researches point out the difference in pressure as well ().Under the conditions of paraneural and extrafascicular application, local anesthetic dilutes quickly into the surrounding tissue and its concentration falls with systemic absorption as well, which results in changes that are of signifi cantly weaker intensity when compared to intrafascicular application; changes are dominantly present in epineurium and in subperineural region of the fasciculus and intensity of the changes depend on the dose and type of local anesthetic (, , , ).Our research shows this distribution of changes during extrafascicular application as well.
Opposite of the topical application, the consequences of the elevation of endoneural pressure during intrafascicular application are edema and ischemia, which results in weakened fl ow in the fasciculus, diffi cult dilution and absorption of local anesthetic and extension of its acting, In cases of intrafascicular application of  lidocaine, we found damages at the level of nerve fi bers, Schwann's cells, intrafascicular blood vessels, interstitium, perineum and epineurium in all nerve samples.Th ose changes were present in the neighboring proximal and distal regions from the application site, but they were of weaker intensity.Other researchers mention similar changes during intraneural application as well, but not in all cases (, ,  and ) because they didn't distinguish between intra-and extrafascicular application, based on the histological nerve structure and the diff erences in achieved application pressures that are in connection with it.

Conclusions
.In intrafascicular application of lidocaine, significantly higher application pressure was found compared to the one in extrafascicular application..Intrafascicularly injected lidocaine results in significant lesions in the puncture area, where subperineural changes are more intensive than central ones.Segments that are distal and proximal to the puncture site show one-way changes of weaker intensity,especially proximally..In extrafascicular application of lidocaine, changes in epineurium and subperineurally located fi bers are dominant.
Figure , , ).Schwann's cells (Figure , ) in the structure of myelin fi bers are hypertrophic and some of them noticeably stick out into the interstitium.Consequently, one gets the impression of their separation from the fi ber structure.At the same time, their cytoplasm and enlarged nuclei have an extraordinary affi nity for the stain.Schwann's cells in the structure of amyelin fi bers have less hyperchromatic nuclei.Apart from the above described changes within the fasciculus, a hypercellularity can be noticed (Figure , ), contributed by both increased number of Schwann's cells and the mobile cells of the connective tissue, especially macrophages.Intrafascicular blood vessels show hypertrophic endothelium (Figure ).Individual or grouped extravasally located erythrocytes can be seen in some areas (Figure ).Epineurium (Figure , ) shows hypercellularity of the mononuclear inflammatory process type, with