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  • Author or Editor: Rachel Sarabia-Estrada x
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Camilo A. Molina, Rachel Sarabia-Estrada, Ziya L. Gokaslan, Timothy F. Witham, Ali Bydon, Jean-Paul Wolinsky and Daniel M. Sciubba

Object

Recombinant human bone morphogenetic proteins (rhBMPs) are FDA-approved for specific spinal fusion procedures, but their use is contraindicated in spine tumor resection beds because of an unclear interaction between tumor tissue and such growth factors. Interestingly, a number of studies have suggested that BMPs may slow the growth of adenocarcinomas in vitro, and these lesions represent the majority of bony spine tumors. In this study, the authors hypothesized that rhBMP-2 placed in an intraosseous spine tumor in the rat could suppress tumor and delay the onset of paresis in such animals.

Methods

Twenty-six female nude athymic rats were randomized into an experimental group (Group 1) or a positive control group (Group 2). Group 1 (tumor + 15 μg rhBMP-2 sponge, 13 rats) underwent transperitoneal exposure and implantation of breast adenocarcinoma (CRL-1666) into the L-6 spine segment, followed by the implantation of a bovine collagen sponge impregnated with 15 μg of rhBMP-2. Group 2 (tumor + 0.9% NaCl sponge, 13 rats) underwent transperitoneal exposure and tumor implantation in the lumbar spine but no local treatment with rhBMP-2. An additional 8 animals were randomized into 2 negative control groups (Groups 3 and 4). Group 3 (15 μg rhBMP-2 sponge, 4 rats) and Group 4 (0.9% NaCl sponge, 4 rats) underwent transperitoneal exposure of the lumbar spine along with the implantation of rhBMP-2– and saline-impregnated bovine collagen sponges, respectively. Neither of the negative control groups was implanted with tumor. The Basso-Beattie-Bresnahan (BBB) scale was used to monitor daily motor function regression and the time to paresis (BBB score ≤ 7).

Results

In comparison with the positive control animals (Group 2), the experimental animals (Group 1) had statistically significant longer mean (25.8 ± 12.2 vs 13 ± 1.4 days, p ≤ 0.001) and median (20 vs 13 days) times to paresis. In addition, the median survival time was significantly longer in the experimental animals (20 vs 13.5 days, p ≤ 0.0001). Histopathological analysis demonstrated bone growth and tumor inhibition in the experimental animals, whereas bone destruction and cord compression were observed in the positive control animals. Neither of the negative control groups (Groups 3 and 4) demonstrated any evidence of neurological deterioration, morbidity, or cord compromise on either gross or histological analysis.

Conclusions

This study shows that the local administration of rhBMP-2 (15 μg, 10 μl of 1.5-mg/ml solution) in a rat spine tumor model of breast cancer not only fails to stimulate local tumor growth, but also decreases local tumor growth and delays the onset of paresis in rats. This preclinical experiment is the first to show that the local placement of rhBMP-2 in a spine tumor bed may slow tumor progression and delay associated neurological decline.

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Rachel Sarabia-Estrada, Jacinto Bañuelos-Pineda, Laura P. Osuna Carrasco, Salvador Jiménez-Vallejo, Ismael Jiménez-Estrada, Efrain Rivas-Celis, Judith M. Dueñas-Jiménez and Sergio H. Dueñas-Jiménez

OBJECT

Transection of peripheral nerves produces loss of sensory and/or motor function. After complete nerve cutting, the distal and proximal segment ends retract, but if both ends are bridged with unaltered chitosan, progesterone-impregnated chitosan, or silicone tubes, an axonal repair process begins. Progesterone promotes nerve repair and has neuroprotective effects thwarting regulation of neuron survival, inflammation, and edema. It also modulates aberrant axonal sprouting and demyelination. The authors compared the efficacy of nerve recovery after implantation of progesterone-loaded chitosan, unaltered chitosan, or silicone tubes after sciatic nerve transection in rats.

METHODS

After surgical removal of a 5-mm segment of the proximal sciatic nerve, rats were implanted with progesterone-loaded chitosan, unaltered chitosan, or silicone tubes in the transected nerve for evaluating progesterone and chitosan effects on sciatic nerve repair and ipsilateral hindlimb kinematic function, as well as on gastrocnemius electro-myographic responses. In some experiments, tube implantation was performed 90 minutes after nerve transection.

RESULTS

At 90 days after sciatic nerve transection and tube implantation, rats with progesterone-loaded chitosan tubes showed knee angular displacement recovery and better outcomes for step length, velocity of locomotion, and normal hindlimb raising above the ground. In contrast, rats with chitosan-only tubes showed reduced normal raising and pendulum-like hindlimb movements. Aberrant fibers coming from the tibial nerve innervated the gastrocnemius muscle, producing electromyographic responses. Electrical responses in the gastrocnemius muscle produced by sciatic nerve stimulation occurred only when the distal nerve segment was stimulated; they were absent when the proximal or intratubular segment was stimulated. A clear sciatic nerve morphology with some myelinated fiber fascicles appeared in the tube section in rats with progesterone-impregnated chitosan tubes. Some gastrocnemius efferent fibers were partially repaired 90 days after nerve resection. The better outcome in knee angle displacement may be partially attributable to the aberrant neuromuscular synaptic effects, since nerve conduction in the gastrocnemius muscle could be blocked in the progesterone-impregnated chitosan tubes. In addition, in the region of the gap produced by the nerve resection, the number of axons and amount of myelination were reduced in the sciatic nerve implanted with chitosan, progesterone-loaded chitosan, and silicone tubes. At 180 days after sciatic nerve sectioning, the knee kinematic function recovered to a level observed in control rats of a similar age. In rats with progesterone-loaded chitosan tubes, stimulation of the proximal and intratubular sciatic nerve segments produced an electromyographic response. The axon morphology of the proximal and intratubular segments of the sciatic nerve resembled that of the contralateral nontransected nerve.

CONCLUSIONS

Progesterone-impregnated chitosan tubes produced aberrant innervation of the gastrocnemius muscle, which allowed partial recovery of gait locomotion and could be adequate for reinnervating synergistic denervated muscles while a parent innervation is reestablished. Hindlimb kinematic parameters differed between younger (those at 90 days) and older (those at 180 days) rats.

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Patricia Zadnik, Rachel Sarabia-Estrada, Mari L. Groves, Camilo Molina, Christopher Jackson, Edward McCarthy, Ziya L. Gokaslan, Ali Bydon, Jean-Paul Wolinsky, Timothy F. Witham and Daniel M. Sciubba

Object

Metastatic spine disease is prevalent in cancer victims; 10%–30% of the 1.2 million new patients diagnosed with cancer in the US exhibit spinal metastases. Unfortunately, treatments are limited for these patients, as disseminated disease is often refractory to chemotherapy and is difficult to treat with surgical intervention alone. New animal models that accurately recapitulate the human disease process are needed to study the behavior of metastases in real time.

Methods

In this study the authors report on a cell line that reliably generates bony metastases following intracardiac injection and can be tracked in real time using optical bioluminescence imaging. This line, RBC3, was derived from a metastatic breast adenocarcinoma lesion arising in the osseous spine of a rat following intracardiac injection of MDA-231 human breast cancer cells.

Results

Upon culture and reinjection of RBC3, a statistically significantly increased systemic burden of metastatic tumor was noted. The resultant spine lesions were osteolytic, as demonstrated by small animal CT scanning.

Conclusions

This cell line generates spinal metastases that can be tracked in real time and may serve as a useful tool in the study of metastatic disease in the spine.

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Patricia L. Zadnik, Camilo A. Molina, Rachel Sarabia-Estrada, Mari L. Groves, Michele Wabler, Jana Mihalic, Edward F. McCarthy, Ziya L. Gokaslan, Robert Ivkov and Daniel Sciubba

Object

The goal of this study was to optimize local delivery of magnetic nanoparticles in a rat model of metastatic breast cancer in the spine for tumor hyperthermia while minimizing systemic exposure.

Methods

A syngeneic mammary adenocarcinoma was implanted into the L-6 vertebral body of 69 female Fischer rats. Suspensions of 100-nm starch-coated iron oxide magnetic nanoparticles (micromod Partikeltechnologie GmbH) were injected into tumors 9 or 13 days after implantation. For nanoparticle distribution studies, tissues were harvested from a cohort of 36 rats, and inductively coupled plasma mass spectrometry and histopathological studies with Prussian blue staining were used to analyze the samples. Intratumor heating was tested in 4 anesthetized animals with a 20-minute exposure to an alternating magnetic field (AMF) at a frequency of 150 kHz and an amplitude of 48 kA/m or 63.3 kA/m. Intratumor and rectal temperatures were measured, and functional assessments of AMF-exposed animals and histopathological studies of heated tumor samples were examined. Rectal temperatures alone were tested in a cohort of 29 rats during AMF exposure with or without nanoparticle administration. Animal studies were completed in accordance with the protocols of the University Animal Care and Use Committee.

Results

Nanoparticles remained within the tumor mass within 3 hours of injection and migrated into the bone at 6, 12, and 24 hours. Subarachnoid accumulation of nanoparticles was noted at 48 hours. No evidence of lymphoreticular nanoparticle exposure was found on histological investigation or via inductively coupled plasma mass spectrometry. The mean intratumor temperatures were 43.2°C and 40.6°C on exposure to 63.3 kA/m and 48 kA/m, respectively, with histological evidence of necrosis. All animals were ambulatory at 24 hours after treatment with no evidence of neurological dysfunction.

Conclusions

Locally delivered magnetic nanoparticles activated by an AMF can generate hyperthermia in spinal tumors without accumulating in the lymphoreticular system and without damaging the spinal cord, thereby limiting neurological dysfunction and minimizing systemic exposure. Magnetic nanoparticle hyperthermia may be a viable option for palliative therapy of spinal tumors.

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Rachel Sarabia-Estrada, Alejandro Ruiz-Valls, Sagar R. Shah, A. Karim Ahmed, Alvaro A. Ordonez, Fausto J. Rodriguez, Hugo Guerrero-Cazares, Ismael Jimenez-Estrada, Esteban Velarde, Betty Tyler, Yuxin Li, Neil A. Phillips, C. Rory Goodwin, Rory J. Petteys, Sanjay K. Jain, Gary L. Gallia, Ziya L. Gokaslan, Alfredo Quinones-Hinojosa and Daniel M. Sciubba

OBJECTIVE

Chordoma is a slow-growing, locally aggressive cancer that is minimally responsive to conventional chemotherapy and radiotherapy and has high local recurrence rates after resection. Currently, there are no rodent models of spinal chordoma. In the present study, the authors sought to develop and characterize an orthotopic model of human chordoma in an immunocompromised rat.

METHODS

Thirty-four immunocompromised rats were randomly allocated to 4 study groups; 22 of the 34 rats were engrafted in the lumbar spine with human chordoma. The groups were as follows: UCH1 tumor–engrafted (n = 11), JHC7 tumor–engrafted (n = 11), sham surgery (n = 6), and intact control (n = 6) rats. Neurological impairment of rats due to tumor growth was evaluated using open field and locomotion gait analysis; pain response was evaluated using mechanical or thermal paw stimulation. Cone beam CT (CBCT), MRI, and nanoScan PET/CT were performed to evaluate bony changes due to tumor growth. On Day 550, rats were killed and spines were processed for H & E–based histological examination and immunohistochemistry for brachyury, S100β, and cytokeratin.

RESULTS

The spine tumors displayed typical chordoma morphology, that is, physaliferous cells filled with vacuolated cytoplasm of mucoid matrix. Brachyury immunoreactivity was confirmed by immunostaining, in which samples from tumor-engrafted rats showed a strong nuclear signal. Sclerotic lesions in the vertebral body of rats in the UCH1 and JHC7 groups were observed on CBCT. Tumor growth was confirmed using contrast-enhanced MRI. In UCH1 rats, large tumors were observed growing from the vertebral body. JHC7 chordoma–engrafted rats showed smaller tumors confined to the bone periphery compared with UCH1 chordoma–engrafted rats. Locomotion analysis showed a disruption in the normal gait pattern, with an increase in the step length and duration of the gait in tumor-engrafted rats. The distance traveled and the speed of rats in the open field test was significantly reduced in the UCH1 and JHC7 tumor–engrafted rats compared with controls. Nociceptive response to a mechanical stimulus showed a significant (p < 0.001) increase in the paw withdrawal threshold (mechanical hypalgesia). In contrast, the paw withdrawal response to a thermal stimulus decreased significantly (p < 0.05) in tumor-engrafted rats.

CONCLUSIONS

The authors developed an orthotopic human chordoma model in rats. Rats were followed for 550 days using imaging techniques, including MRI, CBCT, and nanoScan PET/CT, to evaluate lesion progression and bony integrity. Nociceptive evaluations and locomotion analysis were performed during follow-up. This model reproduces cardinal signs, such as locomotor and sensory deficits, similar to those observed clinically in human patients. To the authors’ knowledge, this is the first spine rodent model of human chordoma. Its use and further study will be essential for pathophysiology research and the development of new therapeutic strategies.