Large naturally-produced electric currents and voltage traverse damaged mammalian spinal cord
Immediately after damage to the nervous system, a cascade of physical, physiological, and anatomical events lead to the collapse of neuronal function and often death. This progression of injury processes is called "secondary injury." In the spinal cord and brain, this loss in function and anatomy is largely irreversible, except at the earliest stages. We investigated the most ignored and earliest component of secondary injury. Large bioelectric currents immediately enter damaged cells and tissues of guinea pig spinal cords. The driving force behind these currents is the potential difference of adjacent intact cell membranes. For perhaps days, it is the biophysical events caused by trauma that predominate in the early biology of neurotrauma.
An enormous (≤ mA/cm2) bioelectric current transverses the site of injury to the mammalian spinal cord. This endogenous current declines with time and with distance from the local site of injury but eventually maintains a much lower but stable value (< 50 μA/cm2).
The calcium component of this net current, about 2.0 pmoles/cm2/sec entering the site of damage for a minimum of an hour, is significant. Curiously, injury currents entering the ventral portion of the spinal cord may be as high as 10 fold greater than those entering the dorsal surface, and there is little difference in the magnitude of currents associated with crush injuries compared to cord transection. Physiological measurements were performed with non-invasive sensors: one and two-dimensional extracellular vibrating electrodes in real time. The calcium measurement was performed with a self-referencing calcium selective electrode.
The enormous bioelectric current, carried in part by free calcium, is the major initiator of secondary injury processes and causes significant damage after breach of the membranes of vulnerable cells adjacent to the injury site. The large intra-cellular voltages, polarized along the length of axons in particular, are believed to be associated with zones of organelle death, distortion, and asymmetry observed in acutely injured nerve fibers. These data enlarge our understanding of secondary mechanisms and provide new ways to consider interfering with this catabolic and progressive loss of tissue.
KeywordsSpinal Cord Spinal Cord Injury Injury Site Secondary Injury Krebs Solution
It is the early events following severe injury to the brain and spinal cord that have received significant attention. In part this is to better understand the progression of tissue damage and to develop means to interfere with it. Many factors have been considered to play a role in the collapse of the spinal cord and brain architecture within hours to days post-injury. This period of time, variable in extent, is usually referred to as "secondary injury" , the primary injury being the acute mechanical insult to the tissue. The biology/pathology forming the basis for secondary injury in the mammalian CNS includes – but is not limited to – particular biochemistries such as: the formation of reactive oxygen species (so-called free radicals) and the initiation of lipid peroxidation of the inner membrane which begins immediately after mechanical damage to CNS cells [2, 3] the formation of endogenous toxins that accumulate within damaged neurons and their processes ; the loss of myelin and the associated collapse of electrophysiological conduction [1, 4]; and the initiation of both apopotosis and progressive necrosis by chemically-mediated events. These are the two main forms of cell death in adult animals, and each plays a role in the demise of CNS parenchyma after mechanical damage [3, 5].
The role of the endogenous bioelectric (ionic) current and the generation of steady DC voltages within damaged CNS parenchyma have been largely ignored. The usual review and narrative of the progression of secondary injury mechanisms begins with the mention of calcium entry into the cytoplasm of cells and their processes (in the special case of glia and neurons). Likewise it is left to the reader to surmise what mechanisms lead to the influx of Ca2+ . In fact it is assumed that the majority of ions in the extracellular milieu – Na+ and Ca2+ which enter the cell, and K+ which leaves it – are a simple matter of diffusion down their concentration gradients. Of course this is an electrochemical gradient, but there are other forces at work that drive the initial biophysical and electrophysiological process which initiate and prolong the progression of secondary injury.
Here we present the first non-invasive measurements of very large (≤ mA/cm2) electric currents driven into the site of damage in fully adult mammalian (guinea pig) spinal cords. This is a true DC electrical current, carried by ions, that is driven by the Electromotive Force (EMF) of the surrounding intact cell membranes. While this electrical injury (both current and voltage mediated) cannot be separated from the earliest pathophysiology associated with mechanical injury, neither can they be ignored. We discuss the role such huge levels of electric current may play in the responses of nerve cells to damage, in particular, the ionic components of this ionic/bioelectric current and possible means to interfere with it.
Peak currents and their dynamics
Modeling instantaneous injury currents and the definition of dorsal and ventral differences in them
One potentially important observation was the apparent dominance of injury currents entering the ventral surface of the spinal cord relative to those measured from the dorsal surface. To more keenly understand and verify these observations required an improvement in our extrapolation of current densities at the surface of the cord, at the instant of injury, and at times after that. This was necessitated by the desire to compare these different regions of the injury in the spinal cord.
Peak spinal cord injury currents measured on ventral and dorsal portion of guinea pig spinal cords.
Vertical portion of spinal cord (μA/cm2)
Dorsal portion of spinal cord (μA/cm2)
The "battery" (EMF) driving this flow of current into axons is the inwardly negative potential (~50 – 70 mV) across undamaged cellular membrane at variable distances from the site of mechanical damage. It is convenient to consider that the inwardly negative membrane potential(s) "short-circuit" ionic current through the compromised integrity (hence significantly reduced resistance) of damaged membranes, producing trans-axonal DC current flow (reviewed in ).
A rapid decay of initial current density was expected and had been reported in measurements made from complete transection of individual giant reticulospinal axons in the ammocoete lamprey . Even given the striking anatomical differences between this proto-vertebrate model, possessing 40–60 μm diameter unmyelinated axons, and the mammal, the dynamics of current decline were remarkably similar. Initial densities were on the order of less than 1 mA/cm2 entering lamprey giant axons and nearby parenchyma, reaching stable densities in the tens of μA/cm2. The ammocoete lamprey's entire brain and spinal cord can be removed and maintained for up to a week in organ culture since the ammocoete's CNS is not intrinsically vascularized. These facts permitted measurements of the injury current entering the transection for many days after injury in the lamprey but cannot be done with the mammalian spinal cord ex vivo. Based on the similarities of these data, it is reasonable to expect the plateau current in the mammalian cord is also likely to persist for many days post-injury
Geometric and tissue considerations
The net current densities reported here are the sum of all internally directed cellular injury currents minus any outwardly directed current that may be produced by the epithelial-like investments of the spinal cord. Epithelial driven ionic currents will be outwardly directed (usually abluminal) while cellular injury currents are driven into damaged cells. It is the net current, i.e. the ions that carry the current into cytosol, that is the prime culprit in the process of early secondary injury. It has been reported that both types of injury currents can occur simultaneously and influence the dynamics of the net current flow into cells, for example, the outflow of "stump currents" subsequent to the amputation of salamander limbs , and in the case of streaming potentials (injury current) in mammalian bone .
The elongate geometry and parallel arrangement of the axons of mammalian white matter supports the dynamics of a strong and steady ionic current flow through and along the long axis of the spinal cord. Injury to axons close to the cell body usually results in death of the cell . This death is related to the amount of calcium influx and the distance that it penetrates along the axon as Ca2+ invades the cytosol. The closer to the cell body that the axon is injured, the greater the chance that the entire cell will succumb to the injury. In white matter long tracts, the site of damage is often very far from the cell bodies giving rise to these fibers, thus the cell and the proximal segment of fiber remain viable until the spontaneous sealing of the axonal membrane. The resistivity of white matter in the long axis of the spinal cord (300 – 400 Ω cm) – is 4–5 fold smaller than the resistivity measured in the transverse direction (1500 – 2500 Ω cm). Thus, mammalian spinal cord white matter is strongly anisotropic in terms of its electrical conductance in the longitudinal and transverse axis. This ensures a preferential circuit along and not across the axons making up the tissue, further suggesting a reduced loss in the peak magnitudes of longitudinal current by tangential current flow in white matter. Furthermore, this anisotropy would be expected to support a standing DC voltage gradient in the long axis of the cord, inside and outside of the axons that make it up. This voltage would be internally positive at the site of the injury and less positive (i.e. more negative) at distances from the point of injury in the tissue of the spinal cord. This is true no matter what types of axons are considered, as the steady DC voltage gradient will be expressed in this polarity independent of the direction of impulse conduction, i.e. in ascending or descending white matter tracts (see below).
Ionic current entering the injury site is carried by the ions most concentrated in the external medium [7, 11]. This would be Na+, Cl- and Ca2+. Of particular interest is the Ca2+ component of this current, since the enormous elevation in cytosolic Ca2+ is correlated to the complete collapse of the cytoarchitecture of axons at the point of damage in addition to facilitating, as a necessary co-factor, many catabolic biochemical cascades ending in necrosis and apoptosis. This role in the catabolism/destruction of cells from outside cytosolic invaders has been well understood since the pioneering work of William Schlaepher and Richard Bunge .
We have emphasized the role of the calcium ion; however this is only in recognition of the importance given it in this literature. In fact, calcium is only one of several key players in the electrical/ionic disturbance after mechanical injury. Increase in cytosolic Na+ also induces a secondary rise in Ca2+, as this triggers Ca2+ release from intracellular stores. This role of Na+ initiated Ca2+ release has also been well described since the seminal studies of Carafoli and Crompton  but is usually largely ignored. This elevation in intracellular Ca2+ is thus expressed as a gradient itself: high at the point of damage and falling with distance (hence the potential difference) along the long axis of the axon. Using a calcium specific ion selective electrode, we have measured about 1.9 – 2 pmoles/cm2/sec of free calcium entering the site of damage. In such an electrode, a Liquid Ion Exchange (LIE) based microelectrode is the sensor, rather than the platinum black tipped probe. Given that the intracellular concentration of calcium is on the order of nmolar – tenths of μ molar, we suspect again that the lessons learned in fish giant axons may be true here as well: that a calcium gradient in the axoplasm dominates and at saturation levels near the site of injury to cells and tissues. Note our measurements in the mammalian cord do not show much of a decrease in Ca2+ entry at the injury site over the first hour, while the net current falls in magnitude over this time. This influx of Ca2+ is of course associated with the complete destruction of cytoarchitecture but may have more subtle effects. This might also indicate that the diffusion dependent element of the current is composed of primarily an influx of Na2+, Cl-, Ca2+ and efflux of K+ and is the major contributor to the large initial net injury current. The plateau current, however, indicates the presence of the driving EMF developed by the adjacent undamaged cell membranes which continue to drive this smaller component into the insult. This keeps driving an ionic front of which Ca2+ is a major part even hours after the initial injury along the initially uninjured portions of the spinal cord. This inevitably leads to spread of the injury. This notion is supported by the observation that the Ca2+ current even 60 mins post injury is not much different from that observed immediately post injury.
We believe the fall in potential along the axis of the spinal cord from the injury site associated with the fall in free calcium may likely be correlated to the zones of cytoplasmic and organelle disruption that extend themselves along the long axis of the axon [14, 15]. Furthermore, this gradient in free calcium has been visualized in the tips of severed Lamprey giant axons using the florescent probe for Ca2+ (FURA II) . The distal to proximal fall in the voltage associated with injury currents within the axon includes zones of peculiar organelle derangements with distance from the disruption, particularly mitochondrial rearrangement. In this zone of damage, mitochondria are arranged in "chains," with their long axis aligned with the length of the axon [14, 15]. We believe this may be an expression of voltage mediated effects of ionic current flow through the nerve fiber, acting to impose a preferred orientation on charged components (organelles and pieces of organelles) within the electrical field associated with the intra-axonal responses to the ions (principally Ca2+ and Na+) that carry the current.
Finally the exodus of K+ is singularly competent to shut down the conduction of nerve impulses in locally damaged axons and thus contributes to the total physiological and behavioral deficit observed immediately after acute CNS damage.
Dorsal vs. ventral
The difference between dorsal and ventral injury currents might have been expected, having its root in the anatomical differences in mammalian white matter of these regions. The dorsal half of ventral white matter, closest to the ventral horns of gray matter, contains mainly small- and medium-diameter axons. In contrast, more ventral regions possess similar caliber spectra – but also comparatively large-diameter axons – approaching or exceeding 10 μm in diameter . While membrane voltages collapse at or very near the actual site of mechanical damage at distances from this region, a significant EMF is maintained across axolemmas by the physiological pumps residing in all normal membranes. Thus a greater surface area of membrane associated with greater numbers of large caliber axons (indeed the "batteries in series") may likely relate to the observed differences between ventral and dorsal white matter. In the same breath, we admit that this answer is hypothetical and may only be a partial explanation among several. For example, one could surmise that the resting potential of dorsal axolemmas might be lower (hence the EMF supporting the injury current) though we do not know of any physiological measurements suggesting this.
The general phenomenology of large magnitude and significant injury current entering spinal cords could be predicted from classical studies of injury potential or demarcation currents from the mid-nineteenth century to the early part of the 20th century by the discoverer of the Action Potential (Emil DuBoise-Reymond; 1818 – 1896) and other 19th century physiologists. These voltages, sampled with galvanonmeters, were extracellularly negative at the site of damage relative to positions farther away and were independent of the conduction pathway. This suggested an axial current flow in nerves produced by injury that appeared to contradict the orthodromic propagation of action potentials in a preferred direction. This quandary was rejected by the influential developmentalist Paul Weiss (1898 – 1989) who strongly condemned "demarcation potentials" as measurement artifacts and also condemned the early observations of preferential direction of growth of neurites in culture to an applied voltage gradient, after failing to duplicate them . As it turned out, Weiss got it completely wrong on both counts. Raphael Lorente de No', the student of Santiago Ramon y Cahal in Madrid, wrote in 1947 "It appears now that Weiss's explanation was erroneous and therefore the observation of the classical authors were significant, even though they cannot be regarded as a proof that an axial current flows in nerve." (, page 92; he was addressing the normal state of an undamaged nerve). Lorente de No's continued neurophysiological studies brought him to the understanding that the failure of action potentials at a position very near to the end of a severed segment of nerve was related to the decreased polarization of membrane, associated with the "demarcation potential" in his view. He wrote: "The explanation of the phenomenon must rather be based on the circumstances attending the decrease in the demarcation current that had been produced by the injury" , page 450]... It is thinkable; therefore, that the continued flow of the demarcation current into the last few millimeters of a regenerating nerve is a mechanism by means of which energy is transferred to the regenerating end from points at some distance from it." (, Page 459; he is discussing axonal regeneration). These issues and controversies evaporated with the birth of the microelectrode age a decade later; however, they bear special recognition here.
Finally we note the now well established clinical use of applied DC voltages arranged parallel with the orientation of white matter in severely injured human spinal cords [1, 19, 20, 21]. One suggested mechanism of action underlying the preservation of anatomy and subsequently behavioral recovery is a reduction in retrograde degeneration in nervous tissue in response to distally negative gradients of applied DC voltage [1, 11]. It is unlikely that an artificially imposed voltage could be used as a therapy in the first minutes after an injury as it is now being used in severe acute spinal cord injury days later . However our findings should reinvigorate the possibility of using calcium blockers if they could be safely administered for a very short time at the site of an accident.
A very large (≤ 1.0 mA) bioelectric current enters the region of damage in the mammalian spinal cord. It is driven by intact "battery" of cell membranes in undamaged adjacent regions. This magnitude of current is similar in both cut and crushed spinal cords. The magnitude falls rapidly by more than an order of magnitude within minutes of the injury. This ionic current is related to the catastrophic destruction of the anatomy of crushed and cut fibers, extending away from the local site of the insult. Particularly important is the calcium component of the current which enters in concentrations of ~2 picmoles of Ca+2 per second per square centimeter. Increases in ionic Ca+2 above its physiological range is related to the destruction of cell architecture and the enabling of catabolic enzymes in the cytosol as an obligatory cofactor. Curiously, levels of current entering the ventral region of the spinal cord were greater than the injury current entering the dorsal regions of crushed spinal cords. Interfering with the Ca+2 mediated destruction dependent on the ionic current of injury in the early acute phase of the injury might be considered as a means of ameliorating the effects of secondary injury.
Materials and methods
Isolation of the spinal cord
Guinea pig spinal cords were isolated using previously specified techniques [22, 23, 24]. Ketamine (80 mg/kg), xylazine(12 mg/kg), and acepromazine(0.8 mg/kg) were used to anaesthetize adult guinea (350–500 gms). The guinea pig hearts were then perfused with 500 ml of oxygenated Krebs solution [124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 2 mM CaCl2, 20 mM dextrose, 26 mM NaHCO3 and 10 mM sodium ascorbate], equilibrated with 95% O2 and 5% CO2 to remove blood and lower the body temperature. The vertebral column was excised, spinal cords quickly removed and immersed in cold Krebs solution. All animal use received prior approval by the Purdue University Animal Care and Use committee, in strict accordance with Federal, State, and University guidelines.
Handling and compression of spinal cords
The ~35 – 40 mm long spinal cords were kept at a room temperature until use and the Krebs solution was replaced every 20 minutes. A 60 mm silicone polymer (sylgard) bottomed petri dish was used to mount the spinal cord for electrophysiological recordings. Stainless steel minutien pins (0.1 mm) obtained from Fine Science Tools (Foster City, CA) were used to carefully pin down the spinal cord at its ends. The crush/compression injury was made with a laboratory – fabricated forceps possessing a détente to help standardize the extent of compression between cords . All spinal cord injuries were timed using a stop watch and the time that elapsed between the injury and the actual recording of the data was subsequently recorded. A constant perfusion was provided in the petri dish to ensure a continuous supply of fresh Krebs solution to the spinal cord while the experiments were being performed.
Vibrating electrodes for the measurement of extracellular current
Measurements were made with non- invasive one dimensional (1 D) and neutating (or 2 D) probes for the measurement of extracellular current [7, 26, 27]. The former gives the density of electric current entering or leaving a biological source normal to its surface with time, while the latter provides this as well as two-dimensional information in the form of current density vectors. Spatial resolution is on the order of 20 μm, and, depending on the resistivity of the bathing media, such probes can detect current densities on the order of picoA/cm2 – far below the resolution required here. Current Vectors are displayed as raw data by software and are superimposed over the digital video image and captured by digital image acquisition.
Microelectrodes used for fabricating the vibrating probes were Pt/Ir electrodes (Micro Probe Inc, Gaithersburg, MD) with a 3 – 5 μm exposed tip, while the rest of the electrode was insulated. The tip of the probe was platinum blackened by electroplating to form a 25–30 μm diameter Pt ball. Alternately, the platinum tip can be replaced with one of a calcium specific resin, which then measures only the calcium component of the net current flow. The completed probes were then calibrated in Krebs solution at 37 degrees C as were physiological measurements. A KCl filled glass microelectrode was used as a point source for calibrations (see below). The point source was made using a 1.5 mm internal diameter borosilicate glass capillary tube pulled to a tip diameter of 8–10 μm. This was performed on a David Kopf Vertical puller (David Kopf Instruments, Tujunga, CA). The probe was vibrated at a distance of one tip diameter between its two extreme positions with X and Y frequencies ranging from 250–300 Hz. The probe actually measures the small voltage difference between its extreme positions with a phase/frequency lockin amplifier. This voltage difference together with the known resistivity of the media is used to calculate the bulk current or the current density associated with the sample of interest. The direction of the current vectors shows whether the current is an influx or efflux.
Temporal and spatial profiles of spinal cord injury currents
Using the vibrating voltage probe to study spinal cord injury we measured a large inwardly-directed injury current at the lesion soon after injury to the spinal cord. This current then decreased rapidly in magnitude to approximately 20% of its original magnitude within 30 minutes (refer to Results above). This decay can be approximated by a 3-parameter exponential decay model:
y(t) = y 0 + a exp(-bt)
where y(t) = current density drop as a function of time, t = time, and y0, a and b are empirically derived, normalized constants.
This expression is the basis of the model for all time-dependent current density measurements.
In a separate study, the vibrating probe was brought to a starting position 50 μm away from the surface of the injury site of the spinal cord. The vibrating electrode was then sequentially stepped back away from the injury site at fixed intervals with the injury current density measured at each point. This step-back, or fall-off, profile provides a reasonable assessment of the spatial profiles of the external electrical field associated with the injury to the spinal cord. This would not be expected to be the same as that data taken from a point source, given the complex and extended geometry of the tissue surface.
An exponential linear combination current decay model provided the best fit for these step-back experiments. We used the formula:
y(x) = y 0 + c exp(-dx) + ex
where y(x) = current density drop as a function of distance from injury site, x = distance from injury site, and y0, c, d and e are empirically derived normalized constants. This formula does not account for the current decay with respect to time. To correct for this we applied a correction factor which compensates for this loss to yield the following model;
y(x) + Δy = y 0 + c exp(-dx) + ex + |-ab exp(-bt)Δt|
Finally, it is of interest to know the magnitude of the current entering the spinal cord at the "instant" of injury (time = 0), and to more properly account for the increased magnitude of the current at the surface of the cord from that actually recorded at the standard measurement position. Given the rapid decline in current with time after the acute injury, and the fact that the probe can not be vibrated any closer than 30–50 μm from the cord's surface without damage, this required some separate study and quantitative normalization of the recorded data.
Calculation of the correction for the current "fall off" due to both time and distance is calculated based on the combination of the formulas presented above in Methods, and is as follows:
ΔY(x,t) = |[-CD exp(-Dx) + E]Δx| + |-AB exp(-Bt)Δt|
This current when added to the original current (Y) reveals the injury current at the surface instantaneously after injury for any one experiment:
Y(x,t) = Y + ΔY(x,t) and,
Y(x,t) = Y + |[-CD exp(-Dx) + E]Δx| + |-AB exp(-Bt)Δt|
This derived current density data should not be considered to provide a perfectly accurate picture of the dynamics of injury current flow in the spinal cord; however, it provides the most accurate data extant for understanding the immediate decay in physiological currents at any distance from the injury site. Moreover, this method permitted us to both calculate, then compare, the injury current at t = 0 and x = 0 for raw data measured from both ventral and dorsal portions of the spinal cord.
We would gratefully like to thank the support by General Funds from the CPR (State of Indiana HB 1440), and an endowment from Mrs. Mari Hulman George. We appreciate and thank Mr. Gary Leung and Ms. Brandi Butler for the dissection and preparation of the spinal cords used for measurement.
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