Journal of Undergraduate Research
Volume 5, Issue 1 - October 2003

Ferrous Chloride-induced Lesions in Rat Spinal Cord

Maria Morales

ABSTRACT

Pain is one of the most misunderstood aspects of spinal cord injury. Chronic pain can be observed in spinal cord injured patients even with interruption of the spinothalamic pain pathway. In the effort to investigate this pain in the animal model, a sufficient injury to the spinal cord must be developed.

In the cat, ferrous chloride has been used to produce a lesion that parallels the hemorrhagic damage seen in spinal cord injury. This lesion can be used in the rat to investigate chronic pain due to spinal cord injury.

In this experiment, varying concentrations of FeCl2 were used in the rat to lesion the gray matter and the peripheral white matter, the propriospinal region. Laminectomies were performed at T6-T7 and injections of 100, 150, and 200mM concentrations of FeCl2 were used to induce spinal lesions. The 200mM concentration of FeCl2 produced the desired lesion eliminating the gray matter and propriospinal region. This lesion technique can be used to examine the role of the propriospinal neurons in allodyina and hyperalgesia after spinal cord injury.

INTRODUCTION

Spinal cord injury (SCI) causes traumatic and devastating damage to the central nervous system and its processes. With the high incidence of SCI, there is an increased need to alleviate the problem. Various strides have been made in the effort to characterize SCI; however, the full depth of the effects of the injury still remain unclear.

The need for an effective and appropriate model for the investigation of spinal cord injury has led researchers to propose various methods of reproducing a parallel injury in an animal. Current models include in vitro preparations of neurons exposed to toxic substances or unfavorable conditions (Nara et al 1999), as well as in vivo spinal cord preparations subjected to physical insults such as weight drops and injection-induced lesions (Liu et al 1999; Hao et al 1992). The variation in the models is intended to characterize the multitude of primary and secondary injuries that result in SCI.

The secondary neuronal effects that follow spinal cord injury have been implicated in the massive cascade of damage that results (Amar & Levy 1999). An effort to reduce this secondary damage should be directed at its causes: petechial hemorrhage, inflammation, edema, loss of autoregulation, and other mechanisms (Amar & Levy 1999). Hemorrhage has been shown to be a key mediator in the cascade of secondary injury that results from spinal cord injury (Tator and Koyanagi 1997). A proper model for the effects of hemorrhagic damage to the spinal cord is necessary to accurately characterize SCI and investigate its long-term effects such as pain and paralysis.

Pain is one of the most misunderstood aspects of SCI. Patients with spinal cord injuries often experience pain in lower dermatomes where paralysis may be observed. The pain as a result of spinal cord injury can be seen with severe damage to the spinal cord and also with interruption of the spinothalamic pain pathway (Vierck and Light 2000). One would expect that interruption of this pathway would result in no pain transmittance; however, chronic below-level pain is still observed as allodynia and hyperalgesia (Widerstrom-Noga 2003). It has been hypothesized that the propriospinal spinal neurons are involved in the transmittance of the pain in the absence of the spinothalamic pathway. A lesion of this area would be necessary to determine its role in chronic pain due to SCI.

Ferrous chloride-induced lesions in the cat have been shown to accurately reproduce the damaging effects of a spinal hemorrhage (Anderson and Means 1983). The highly controllable nature of this lesion makes it a prime candidate for a model to study pain due to SCI. The lesion size and longitudinal extent can be controlled by altering the volume and concentration of FeCl2 used. Through this manipulation, the lesion can be designed to damage the gray matter and associated propriospinal pathways.

In this experiment, varying concentrations of FeCl2 were used to determine the proper amount to lesion the gray matter and a small amount of surrounding white matter, the propriospinal region, of the rat spinal cord. The fine-tuning of this lesion will allow for behavioral investigations of pain as it relates to the propiospinal region of the spinal cord.

METHODS

Five female Long Evans hooded rats (M2-5, M7) weighing 280-300g were housed in environmentally enriched cages and given ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida.

Ferrous Chloride Solution

Ferrous chloride tetrahydrate was purchased from Sigma-Aldrich. Ferrous chloride solution was made in 100, 150, and 200 mM concentrations in 0.9% saline solution. Saline was bubbled with nitrogen for 15 minutes prior to addition of ferrous chloride. The ferrous chloride solution was bubbled with nitrogen for approximately 30 minutes prior to intraspinal injection to prevent the solution from oxidizing. One intraspinal injection was performed using ferrous chloride solution without nitrogen treatment (M7).

Intraspinal Injection

The animals were anesthetized with subcutaneous injection of Ketamine (27.8 mg/kg), Xylaxine(5.57 mg/kg), and Acepromazine(0.91 mg/kg) mixture. The surgical area was shaved and cleaned with 70% ethanol and betadine. The animals were placed in a stereotaxic device, and dorsal laminectomies were performed at T6-T7. Ferrous chloride solutions of 100 (M5, M7, and M4), 150 (M2), and 200 mM (M3) were injected (2-l) on contralateral sides of the dorsal spinal cord at 400 and 900 microns below the spinal surface using a micropipette. The deep layers of the incision were closed with absorbable sutures (Mylon-4.0) and the skin was closed with stainless steel staples. Saline solution (2-4 cc) was injected subcutaneously to rehydrate the animal and promote recovery.

Histology

Animals were sacrificed at 2 weeks post-surgery by intracardiac perfusion using a 0.9% saline-4% paraformaldehyde fixation sequence. Tissue was dissected with scar tissue intact and immerse-fixed in 4% paraformaldehyde overnight. Spinal cord tissue was then embedded in Polyfin wax and cut in 20 micron sections. Mounted sections were stained for cell bodies and axons.
Lesion size was assessed by observing lesion progression through the cord. The largest extent of damage to the gray matter and peripheral white matter was recorded. The length of the lesion was also measured.

RESULTS

The Ferrous chloride concentrations of 100mM failed to produce a lesion eliminating the entirety of the gray matter (Figure 1). Lesion M4 failed to encompass a large extent of the gray matter. The longitudinal extent of M4 was 1.22mm. Lesion M5 almost eliminated all of the gray matter, however, the dorsal horns remained intact. The surrounding propriospinal region also remained relatively intact. The longitudinal extent of M5 was 3.36mm. Lesion M7 also failed to eliminate the dorsal horn of the gray matter as well as parts of the ventral horns. The longitudinal extent of M7 was 2.88mm. All three lesions were centered at T6-T7.


Figure 1. Outlines of spinal cord sections showing gray matter location and lesion of the gray matter and peripheral area in sections treated with 100mM FeCl2. The representation of the gray matter is masked showing the lesion in the lower layer. Areas of gray matter without the black lower layer indicate intact gray matter.

The lesion produced by a 150mM concentration of FeCl2 (M2) also failed to encompass much of the dorsal and ventral horns as well as the surrounding propriospinal region. The longitudinal extent of the lesion was 4.72mm (Figure 2).

The 200mM concentration of FeCl2 produced a lesion (M3) of the gray matter as well as the propriospinal region. The longitudinal extent of the lesion was 4.680mm centered at T7-T8 (Figure 2).


Figure 2. Outlines of spinal cord sections treated with 150 (M2) and 200mM (M3) FeCl2 showing lesion size and representation of gray matter.

DISCUSSION

A model of hemorrhagic damage to the spinal cord is necessary to accurately characterize SCI and investigate its long-term effects such as pain and paralysis. Hemorrhagic damage to the spinal cord results in secondary neuronal effects that extend the damage to the cord far beyond the initial primary insult (Amar and Levy 1999). The proper hemorrhagic lesion will be invaluable in the assessment of pain due to spinal cord injury in the animal model.

The pain that results from spinal cord injury can be seen in patients with interruption of the spinothalamic pathway (Vierck and Light 2000). Investigations of damage due to SCI have shown that the propriospinal region of the spinal cord may be intimately involved in the development of hyperalgesia after recovery of pain sensation in dermatomes caudal to the injury (Vierck and Light 2000). A lesion of the gray matter and the surrounding white matter that comprises the propriospinal region of the spinal cord would allow for the investigation of hyperalgesia that results from injury of this region.

Ferrous chloride-induced lesions have been shown to be effective in reproducing the effects of hemorrhagic damage to the spinal cord in the cat (Anderson and Means 1983). This lesion can also be used in the rat to produce a highly specific injury that is easily controlled by altering the volume and concentration of the FeCl2.

The lesions produced by the 100mM and 150mM concentrations of FeCl2 are not sufficient for the investigation of the role of the propriospinal region of the white matter. Lesions M2, M4, M5, and M7 fail to damage all of the gray matter and the propriospinal region. Although lesions M4, M5, and M7 are all produced by the same concentration of FeCl2, they are characteristically very different. Lesion M4 is especially different in that its area is drastically reduced in comparison to the other two lesions. In addition, the longitudinal extent of the lesion is far smaller than those of the other two lesions. Lesions M5 and M7 are more similar in size and longitudinal extent, however, neither lesion affects the necessary regions of the spinal cord.

The variation in the lesion size here can be attributed to minor alterations in the method of preparation of FeCl2 solution. The likely explanation for the difference is a change in oxidation state of the FeCl2 when injected into the spinal cord. The solution may not have been sufficiently treated with the nitrogen gas prior to injection or the FeCl2 may have oxidized in solution prior to injection and after removal from the nitrogen gas pump. This shows that particular caution must be used when preparing the solution in order to ensure the condition of the FeCl2 as its activity is highly dependent upon its oxidation state.

The lesion produced by the 200mM concentration of FeCl2 is the appropriate lesion for the investigation of the role of the propriospinal region of the spinal cord in pain transmittance and hyperalgesia after injury. The lesion affects the gray matter and the surrounding white matter, the propriospinal region, while sparing the majority of the peripheral white matter. This lesion can be used in future investigations of the role of the propriospinal region in the transmittance of pain after spinal cord injury.

REFERENCES

Amar AP, Levy ML. Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury. Neurosurgery. 44(5), 1027-1039, 1999.

Anderson DK, Means ED. Lipid peroxidation in spinal cord FeCl2 induction and protection with antioxidants. Neurochemical Pathology. 1, 249-264, 1983.

Hao JX, Watson BD Xu XJ Wiesenfeld-Hallin Z, Seiger A, Sundstrom E. Protective effect of the NMDA antagonist MK-801 on photochemically induced spinal lesions in the rat. Experimental Neurology. 118, 143-152, 1992.

Liu D, Liu J, Wen J. Elevation of hydrogen peroxide after spinal cord injury detected by using the Fenton Reaction. Free Radical Biology & Medicine. 27(3/4), 478-482, 1999.

Nara K, Konno D, Uchida J, Kuichi Y, Oguchi K. Protective effect of nitric oxide against iron-induced neuronal damage. Journal of Neural Transmission. 106, 835-848, 1999.

Tator CH, Koyanagi I. Vascular mechanisms in the pathophysiology of human spinal cord injury. Journal of Neurosurgery. 86(3), 483-492, 1997.

Vierck Jr. CJ, and Light AR. Allodynia and hyperalgesia within dermatomes caudal to a spinal cord injury in primates and rodents. Progress in Brain Research. 129, 411-428, 2000.

Widerstrom-Noga E. Chronic pain and non-painful sensations after spinal cord injury: Is there a relation? Clinical Journal of Pain. 19(1), 39-47, 2003.

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