Neuropathological effects of intracerebral platinum salt injections.

Multiple intracerebral injections of a mixture of platinum salts were made in 9 adult cats and the brains studied by light and electron microscopy at 5-12 days post injection. At the center of the lesions normal cortical architecture was completely replaced by edematous areas containing lipid-laden macrophages and cellular debris. The lesion periphery was characterized by perivascular edema and degenerative changes including cytoplasmic lipid inclusions and vacuolations with selective vulnerability of neurons. Membranous cytoplasmic bodies (MCB), zebra bodies and multiple nucleoli were observed in several cell types. This ultrastructural pattern, mimicked to some extent, that observed following electrical stimulation of brain following chronically implanted platinum and rhodium electrodes. The induction of zebra bodies and MCB, both of which are morphologic features of human neurolipidoses associated with congenital enzyme deficiencies, suggests an inhibitory effect of platinum on brain enzymes. Functional electrical stimulation of brain and other organs is currently being employed in a wide variety of clinical applications (14, 15). A mandatory consideration is that of the long-term effects of the stimuli as well as the electrodes themselves on the tissues involved. The histological effects and mechanisms of tissue damage following chronic application of electrical stimuli to brain have been the subject of several investigations in this laboratory (1, 14-18). Factors contributing to neural damage induced by electrical stimulation include noxious products resulting from electrode dissolution. In vitro studies employing electrochemical (3,9) and scanning electron microscopy (6) techniques have established that erosion of noble metal electrodes occurs, even when passing relatively small stimulation currents. Such electrode dissolution is of particular importance in long term applications of neural prostheses. The present study was initiated to assess the contribution of platinum electrode erosion products to neurla damage following electrical stimulation of brain, specifically to distinguish morphological changes resulting directly from electrode solubilization as opposed to electrical factors. Accordingly, intracerebral injections of graded volumes of platinum salts were made in an attempt to stimulate the presence of platinum electrode dissolution products.

Histopathologic and physiologic effects of chronic implantation of microelectrodes in sacral spinal cord of the cat.

Active microelectrodes were implanted for a period of 2 weeks to 3 months into the sacral spinal cord of 10 male cats in order to test the feasibility and the safety of discrete stimulation of the parasympathetic preganglionic nucleus for future clinical applications of microelectrode technology in micturition control. An array of four 50 microns-diameter iridium microelectrodes was inserted beneath the dura in each cat. At weekly intervals, bladder pressure was measured as hydrostatic pressure on an intraluminal catheter. At the end of the period, histopathology was evaluated with serial transverse epoxy sections. Observations included diffuse and focal axonal degeneration in white matter and possible neuronal loss around the electrode in the gray matter, meningeal ensheathment of the shafts, and occasional aseptic inflammation of tissue and apparent movement of the electrodes after implantation. Increased bladder pressure responses to individually pulsed electrodes located within the sacral parasympathetic nucleus were not consistent, and, surprisingly, at least 2 different sites were also effective. As long as 3 months after implantation, in 2 out of 5 animals, pulsing of electrodes consistently produced micturition. We conclude that while microelectrode implants are feasible, further modifications in electrode design are needed to eliminate movement and inflammation.

MK-801 protects against neuronal injury induced by electrical stimulation.

The ability of MK-801, a non-competitive N-methyl-D-aspartate receptor antagonist, to protect neurons in the cerebral cortex from injury induced by prolonged electrical stimulation was assessed in cats. Platinum disc electrodes 8.0 mm in diameter and with a surface area of 0.5 cm2 were implanted in the subdural space over the parietal cortex. Ten days after implantation of the electrodes, all animals received continuous stimulation for 7 h using charge-balanced, cathodic-first, controlled current pulses with a charge density of 20 microC/cm2 and a charge/phase of 10 microC/phase. They received either no MK-801, or 0.33 or 5.0 mg/kg (i.v.) administered intravenously, just before the start of the stimulation. Immediately following the stimulation, the animals were perfused and the cerebral cortex examined by light microscopy at eight sites beneath the electrodes. Neuronal damage in the form of shrunken, hyperchromic neurons and perineuronal halos was present only beneath the stimulating electrodes; damage was moderate to severe in stimulated animals that had not received MK-801, slight in animals receiving 0.33 mg/kg, and none to slight in animals receiving 5.0 mg/kg. These results indicate that MK-801, in an apparently dose-dependent fashion, provides substantial but not complete protection against neuronal injury induced by prolonged electrical stimulation. Thus prolonged electrical stimulation can be added to the list of neuropathologic conditions which involve glutamate-induced excitotoxic damage via the N-methyl-D-aspartate receptor. The results also support the hypothesis of neuronal hyperactivity as a principal cause of electrically-induced injury in the central nervous system. The implications for design of protocols for functional electrical stimulation are discussed.

Ultrastructural effects of Conray 60 and Pantopaque on ependyma and choroid plexus following intraventricular injections.

Electron microscope evaluation of choroid plexus and ependyma following single cerebral intraventricular injections of Conray 60 and Pantopaque was carried out on 35 rats and 4 dogs. The animals were sacrificed at periods ranging from 1 hour to 4 months. Conray was not detected with the light or electron microscope; however, Pantopaque was presumptively localized as electron-dense masses associated with lipid-like bodies at the ventricular interface of both choroidal and ependymal epithelium. Conray 60 injections consistently induced convulsions in rats. Histological studies demonstrated moderate cellular damage and multilayered proliferation of ependymal epithelium. Morphological damage following Pantopaque was more severe and widespread in both choroid plexus and ependyma suggesting that, of the two agents, Conray may have the greater clinical potential provided that the associated convulsions are controlled by appropriate measures.

Histological evaluation of polyesterimide-insulated gold wires in brain.

Polyesterimide-coated gold wires were implanted subdurally on the parietal cortex of rabbits for 16 weeks. Light microscope examination of the implant sites showed no evidence of toxicity. There was no inflammatory response of the adjacent parenchyma. Gliosis in the molecular or neuronal layers was only slight to moderate. Overall, the polymer demonstrated good biocompatibility at 16 weeks after implantation. Future, long-term biocompatibility studies of this material are indicated, including evaluation of tumourigenic properties.

Tissue response to potential neuroprosthetic materials implanted subdurally.

A histologic study was made of the response of the leptomeninges and underlying cerebral cortex of the cat to subdural implantation of 3 insulating materials (HR605-P, Parylene-C and PI-2555) and a polymeric electrode component (MMA/MAPTAC) for periods of 8 and 16 wk. The tissue reactions were compared with those elicited by the arrays of Dacron mesh matrices, pure platinum controls and by positive controls (Ag-AgCl) known to cause reactions in the brain. Sites beneath the Dacron mesh matrix, pure platinum control implants and beneath all insulating materials implanted for 8 and 16 wk appeared indistinguishable, exhibiting little tissue reaction. All neurons appeared normal. The leptomeninges and cortex beneath the Ag-AgCl implants showed a chronic inflammatory reaction after 8 and 16 wk. Despite varying amounts of oedema, gliosis and ingrowth of connective tissue in the molecular layer, virtually all underlying neurons appeared normal.

Electrical stimulation with Pt electrodes. VII. Dissolution of Pt electrodes during electrical stimulation of the cat cerebral cortex.

Procedures are described for determining trace quantities of Pt released into brain tissue directly beneath cortical surface stimulation electrodes. Implanted electrodes (1.1 mm Pt discs) were stimulated for 4.5 h, 9 h and 36 h (4 X 9 h/day) with balanced biphasic pulses (20 micro C/cm2 or 100 micro C/cm2 per phase, 50 Hz), following which tissue 0-2 mm beneath stimulation electrodes and the encapsulating tissue adherent to electrodes was excised and analyzed for Pt. A time-dependent increase in Pt concentration was observed between 4.5 h (4-20 ng Pt/stimulation site) and 9 h (50-339 ng Pt/site) of stimulation at 100 micro C/cm2 with nearly all of the Pt located in the encapsulating tissue associated with the electrodes. Somewhat less Pt was observed in the 36 h samples, and it was almost equally distributed between the encapsulating tissue of the electrodes and the first millimeter depth of underlying brain tissue. Little or no Pt was found at electrode sites receiving 20 micro C/cm2 pulses. Control brain tissue samples as well as samples of blood, CSF and kidney were negative for Pt. The findings indicate that the rate of Pt dissolution gradually decreases during in vivo stimulation, and that dissolved Pt may slowly move away from stimulation sites, possibly by diffusion or fluid exchange.

Characterization of electrode dissolution products on the high-voltage electron microscope.

Deposits left by electrodes and biocompatibility test specimens implanted in brain or peripheral nerve were characterized by X-ray microprobe analysis, electron diffraction and stereoscopic imaging using a high-voltage electron microscope. Examination of thick (1-micron) sections of neural tissue confirmed that the electron-dense bodies found adjacent to electrode positions consist of elements originating in the implant material (with the exceptions of the S and Se found in association with Ag). These elements have no long-range order, suggesting they are complexed with biological molecules. In some cases the deposits appear to be caused by pulsing the electrode with current, while in other cases the deposits are corroded or abraded from the electrode or are otherwise not associated with the neuroprosthetic functioning of the implant.

A quantitative computer-assisted morphometric analysis of stimulation-induced injury to myelinated fibers in a peripheral nerve.

We describe a computer-assisted morphometric procedure for quantifying acute axonal injury induced in peripheral nerves by prolonged electrical stimulation. The procedure is a two-phase process, with the image analysis implemented via a commercial image analysis program, followed by an automated editing of the morphometric parameters of each object identified by the image analysis software. Both phases are implemented on IBM-compatible personal computers. The custom software counts the number of fibers undergoing early axonal degeneration, using a two-category classification scheme based on the range of myelin cross-sectional area and axonal cross-sectional areas of normal (unstimulated) nerves. When the damaged fibers are counted using this procedure, the correlation between the normalized amplitude of the electric stimulus and the number of degenerating fibers is the same as when the analysis is performed by an experienced histopathologist (R = 0.87) and carries the advantage of being entirely objective. The correlation was higher with a two-category classification (damage/no damage) than when the severity of the damage to each axon was weighted according to the amount of axonal shrinkage. We determined that axons 3.5-9 microm in diameter are the most vulnerable to injury from the electrical stimulation. This has certain implications regarding the mechanism underlying this type of injury.

A microelectrode for delivery of defined charge densities.

A procedure is described for fabricating microelectrodes of platinum-30% iridium, insulated with Epoxylite varnish and having beveled ellipsoidal tips obtained by truncating a conical tip at an angle of 30-50 degrees. The geometric surface area of the ellipsoidal facet is reproducible and easily measured. Using these electrodes, neurons in the cerebral cortex of cats have been activated without damage throughout 24 h or more of continuous stimulation at 15-30 microA and charge densities of 150-300 microC/cm2.phase.

The uptake of delta9-tetrahydrocannabinol in choroid plexus and brain cortex in vitro and in vivo.

[3H]delta9 Tetrahydrocannabinol (delta9-THC) was actively transported by the choroid plexus and cerebral cortical slices of the rabbit when incubated as a BSA-microsuspension in artificial rabbit CSF. The transport system for delta9-THC in choroid plexus had a V max of 174 nmoles/mg tissue/h, approximately 9-fold greater than that observed for cortical slices. In vivo experiments demonstrated a preferential distribution of delta9-THC in choroid plexus at 1 h after intravenous injection. These results indicate that delta9-THC is actively accumulated by choroidal epithelium and may also be transported across the epithelial stroma into the capillary circulation. This suggests that the choroid plexus participates in the regulation of delta9-THC concentration in CSF and indirectly in brain by means of the "sink" function of the CSF.

Considerations for safety in the use of extracranial stimulation for motor evoked potentials.

The possibility of neural damage during extracranial brain stimulation for motor evoked potentials (MEPs) is discussed from the perspective of animal studies in which the stimulating electrodes were in direct contact with the brain. These data indicate that the charge per phase used in most of the extracranial MEP protocols is sufficient to induce neural damage if the stimulation is applied continuously for several hours. However, in most cases dispersion of the stimulus current in the extracranial tissue and skull is probably adequate to attenuate the stimulus charge density at the brain surface to a safe level (less than approximately 40 microC/cm2 X ph). However, the possibility exists that low resistance paths between the stimulating electrode and the brain may give rise to foci of high charge density. The possibility of such focusing may be less with magnetic field than with direct electrical field stimulation. We stress the need for additional animal studies designed to delineate a range of safe stimulus parameters for this particular technique.

Histopathological evaluation of dog sacral nerve after chronic electrical stimulation for micturition.

Histological evaluations of dog sacral nerves were carried out after stimulation for electromicturition with three types of circumneural electrodes. The use of two types of cuff arrays was associated with a marked buildup of connective tissue around the nerve and filling the lumen of the array. Nerves within the first type of cuff array (having diameters approximately that of the nerve they surrounded) were often extruded from the lumen of the cuff. In some cases, this was accompanied by moderate or marked loss of axons. It is not clear whether this phenomenon was the result of the growth of connective tissue within the cuff or tension on the electrical leads. The damage cannot be attributed to the electrical stimulation because nerves enclosed by nonpulsed electrodes showed similar damage. The second type of cuff array used in the study had an oversize lumen. There was often considerable growth of connective tissue within the cuffs, but minimal or no mechanical deformation of the included nerves and minimal loss of axons. Because of sealable lips, extrusion of the nerve from this electrode was impossible. The nerves and arrays both functioned well, and there was minimal, if any, mechanical distortion of the nerves and minimal neural damage. Nerves within a third type of array ("spinal" array) also showed no or minimal damage. The array was implanted easily, and the delicate, springlike nature of the matrix allowed close apposition to nerves of different diameters while avoiding constriction of the nerve.

Evaluation of electrode array material for neural prostheses.

Matrix support materials for brain surface electrodes used in neuroprosthetic applications were evaluated after chronic subdural implantation over the parietal cortex of the cat. Four types of array fabricated with Silastic, Dacron mesh, or platinum wire annuli were implanted for periods ranging from 5 weeks to 1 year. We evaluated the arrays by access resistance measurements and gross and histological observations of the tissue beneath both nonstimulated and stimulated electrodes. A porous type matrix constructed of Dacron mesh proved to be the superior design because of its minimal compression of the cortical surface, facility of handling during implantation and autopsy, and satisfactory electrical characteristics provided by a good electrode-brain interface. (Neurosurgery, 5: 681--686, 1979).

Histological evaluation of neural damage from electrical stimulation: considerations for the selection of parameters for clinical application.

The relationship of charge density per phase, or QD/ph (expressed in units of microcoulombs per cm2 per phase of the charge-balanced wave form), and total charge (QDt) to neural damage has been investigated by light and electron microscopy after surface stimulation of the parietal cortex in normal cats. QD/ph values ranging from 40 to 400 were achieved by varying several stimulus parameters. The least amount of neural damage in this study was observed at QD/ph 40). The extent of neural injury at stimulated sites increased with the charge density and was evident as disruption of cell membranes, intracytoplasmic vacoulation, an increasing glycogen content, the deposition of intracellular calcium hydroxyapatite, and neuronal and astrocytic degeneration. Although individual factors contributing to neural damage are isolated with difficulty, charge density and total charge seem to be predominant among the contributing parameters. In view of these findings, recommendations have been made for the selection of electrical stimulus parameters to be used in central nervous system prostheses.

Ultrastructural observations suggesting apocrine secretion in the choroid plexus: a comparative study.

Ultrastructural studies were carried out on the choroid plexus of eight species including amphibian, reptilian, and mammalian forms with special attention directed toward the phenomenon of apical blebbing in choroid plexus epithelial cells. Choroidal blebs in various stages of formation, release, and maturation were observed in all species examined. Evidence presented by this and previous studies suggests that choroidal and ependymal blebbing represents a physiological significant mechanism by which proteins and/or other molecules enter the cerebrospinal fluid (CSF).

Intracellular calcium deposition in brain following electrical stimulation.

The effects of electrical stimulation of the cat cerebral cortex have been evaluated by light and electron microscopy following a wide variety of stimulation parameters (QD/ph of 10 - 300 muC/cm2/ph). Platinum or rhodium disc electrode arrays were bilaterally implanted subdurally on the parietal cortex and subjected to 36-hour stimulations (9 hrs./day for 4 days). Prominent among the degenerative changes shown by electron microscopy were dense crystalline inclusions that were identified as calcium hydroxyapatite (CHA) crystals by electron diffraction and energy dispersive X-ray analysis. The appearance of intracellular calcification generally paralleled the onset of other degenerative changes in stimulated tissue, including gliosis, mitochondrial swelling, lipid inclusions, degenerating cells, neuronal loss, and phagocytic activity. A preferential deposition of calcium was noted in mitochondria of several cell types and in postsynaptic dendrites. The mechanism of the apparently electroresponsive calcium deposition is obscure; however, a plausible explanation is that increased cyclic AMP levels, known to occur with electrical stimulation of nervous tissue, result in enhanced calcium plasmalemmal permeability.

Stimulation with chronically implanted microelectrodes in the cochlear nucleus of the cat: histologic and physiologic effects.

The effects of several hours of continuous electrical stimulation in the cats' cochlear nucleus with chronically implanted activated iridium microelectrodes was investigated from the changes in the evoked response near the inferior colliculus and also by histologic evaluation of the stimulated tissue. The stimulating microelectrodes had geometric surface areas of 75-500 microns2. They were pulsed continuously for 4 h, at a pulse repetition rate of 200 Hz, using charge-balanced pulse pairs. The charge per phase was 1.8 or 3.6 nC/ph. The animals were sacrificed for histologic evaluation 2 h, or several days later. The only remarkable histologic change resulting from the 4 h of stimulation was some aggregation of lymphocytes at the site of stimulation. However, depression of the electrical excitability of neurons near the sites often persisted for several days after 4 h of stimulation at 3.6 nC/phase. The charge per phase of the stimulus pulse pair was correlated strongly with the depression of excitability, and there was a weaker correlation between the depression and the amplitude of the first phase of voltage transient induced across the electrode-tissue interface. The charge density, calculated from the geometric surface area of the stimulating electrodes, was poorly correlated with the severity of the depression. The findings suggest a means of detecting impending stimulation-induced neural damage while it is still reversible.

Stimulus parameters affecting tissue injury during microstimulation in the cochlear nucleus of the cat.

We investigated the effects of continuous microstimulation in the cats' posteroventral cochlear nucleus, using chronically implanted activated iridium microelectrodes. We examined 51 electrode sites (39 pulsed sites, and 12 unpulsed sites). Seven hours of continuous stimulation at 500 Hz often produced tissue injury near the tips of the pulsed microelectrodes. The damage took the form of a region of vacuolated tissue extending 200 microns or more from the site of the electrode tip. Electron microscope studies showed the vacuoles to be severely edematous segments of myelinated axons. The statistical correlation between the amount of damaged tissue and the charge per phase was large and highly significant (P < 0.0001). When the electrodes were pulsed for 7 h at 500 Hz with charge-balanced biphasic pulse pairs, the threshold for the damage was approximately 3 nC/phase. The damage threshold was not appreciably lower than the stimulation protocol was extended to 35 h (7 h/day for 5 days). In contrast, the threshold for exciting neurons near the microelectrode is approximately 1 nC/phase, as determined by the evoked response recorded in the inferior colliculus. There was little correlation between the severity of the tissue damage and the geometric charge density at the surface of the electrodes, between the damage and amplitude of the cathodic phase of the voltage transient induced across the stimulating electrodes by the stimulus current pulses, or between the damage and the stimulus pulse duration.

Charge density and charge per phase as cofactors in neural injury induced by electrical stimulation.

The possibility of neural injury during prolonged electrical stimulation of the brain imposes some constraints on the use of this technique for therapeutic and experimental applications. Stimulating electrodes of various sizes were used to investigate the interactions of two stimulus parameters, charge density and charge per phase, in determining the threshold of neural injury induced by electrical stimulation. Platinum electrodes ranging in size from 0.002 to 0.5 cm2 were implanted over the parietal cortex of adult cats. Penetrating microelectrodes fabricated from iridium, with surface areas of 65 +/- 3 x 10(-6) cm2 were inserted into the parietal cortex. Ten days after implantation, the electrodes were pulsed continuously for 7h using charge balanced, current regulated, symmetric pulse pairs, 400 microseconds per phase in duration, at a repetition rate of 50 Hz. The animals were perfused immediately after the stimulation for histologic evaluation of the brain tissue subjacent to the electrode sites. The results show that charge density (as measured at the surface of the stimulating electrode), and charge per phase, interact in a synergistic manner to determine the threshold of stimulation-induced neural injury. This interaction occurs over a wide range of both parameters; for charge density from at least 10 to 800 microC/cm2 and, for charge per phase, from at least 0.05 to 5.0 microC per phase. The significance of these findings in elucidating the mechanisms underlying stimulation-induced injury is discussed.