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Systemic effects induced by intralesional injection of ω-conotoxin MVIIC after spinal cord injury in rats

Journal of Venomous Animals and Toxins including Tropical Diseases volume 20, Article number: 15 (2014) Cite this article

Abstract

Background

Calcium channel blockers such as conotoxins have shown a great potential to reduce brain and spinal cord injury. MVIIC neuroprotective effects analyzed in in vitro models of brain and spinal cord ischemia suggest a potential role of this toxin in preventing injury after spinal cord trauma. However, previous clinical studies with MVIIC demonstrated that clinical side effects might limit the usefulness of this drug and there is no research on its systemic effects. Therefore, the present study aimed to investigate the potential toxic effects of MVIIC on organs and to evaluate clinical and blood profiles of rats submitted to spinal cord injury and treated with this marine toxin. Rats were treated with placebo or MVIIC (at doses of 15, 30, 60 or 120 pmol) intralesionally following spinal cord injury. Seven days after the toxin administration, kidney, brain, lung, heart, liver, adrenal, muscles, pancreas, spleen, stomach, and intestine were histopathologically investigated. In addition, blood samples collected from the rats were tested for any hematologic or biochemical changes.

Results

The clinical, hematologic and biochemical evaluation revealed no significant abnormalities in all groups, even in high doses. There was no significant alteration in organs, except for degenerative changes in kidneys at a dose of 120 pmol.

Conclusions

These findings suggest that MVIIC at 15, 30 and 60 pmol are safe for intralesional administration after spinal cord injury and could be further investigated in relation to its neuroprotective effects. However, 120 pmol doses of MVIIC may provoke adverse effects on kidney tissue.

Background

Spinal cord injury (SCI) is a leading cause of permanent disability in young adults [1–3]. At the time of trauma, the primary lesion usually leads to the disruption of axons, neurons and neuroglia cell bodies, resulting in nerve impulses interruption. Afterwards, the secondary neurodegenerative events start and worsen the initial injury. Excessive accumulation of intracellular calcium is a common phenomenon after SCI and it is the most critical step in ionic dysregulation that generates axonal injury and eventual apoptosis or necrosis via an increase in cellular enzymes activation, mitochondrial damage, acidosis, and free radicals production [4–9].

Calcium channel blockers (CCB) have shown great potential in reducing brain and spinal cord injury, by preventing the intense ion influx and, consequently, the secondary injury progression [10, 11]. A wide variety of natural CCB were identified in animal venoms containing neuroactive or neuroprotective peptides, including the conotoxins from Conus snails [12]. Omega-conotoxin MVIIC (MVIIC) is a member of the CCB toxin family constituted by 26 aminoacids [13]. It selectively inhibits the types N (Cav2.1), P, and Q (Cav2.2) voltage-dependent calcium channels (VDCC) that are essential in the release of neurotransmitters [14–16]. In recent years, studies on MVIIC effects have shown that it significantly reduces calcium influx through VDCC in several in vitro models of ischemic brain and spinal injuries [15, 17–21]. Thus, these findings suggest a potential role of MVIIC in preventing secondary injuries after spinal trauma.

However, early clinical experience with MVIIC demonstrates that side effects may limit the usefulness of this class of drugs [22, 23]. Envenomation by Conus toxins is characterized by various symptoms such as intense pain followed by shaking, generalized paresthesia, neuromuscular paralysis and death caused by respiratory failure [23, 24]. No information about the pharmacokinetics and pharmacodynamic characteristics of MVIIC is available. To be possible to evaluate the effects of toxin on SCI, safety should be the overriding principle in the selection for the best dose. The present study was designed to determine, for the first time, the in vivo effects of MVIIC on blood profile and histopathological changes that it may provoke. In this experiment, MVIIC was intralesionally injected due to its peptide nature, so the toxin was directly administered in the central nervous system (CNS), and additionally this route enables local treatment, requiring a smaller amount of toxin with fewer side effects [25, 26].

Methods

Experimental design

Thirty adult male Wistar rats weighting 400 to 450 g were randomly distributed into five groups. Rats were housed in a controlled environment and provided with commercial rodent food and water ad libitum. The Ethics Committee on Animal Experimentation of the Federal University of Minas Gerais (CETEA/UFMG) approved the present study under protocol n. 075/10. All animals experiments followed the recommendations of Guide for the Care and Use of Laboratory Animals of the US National Institute of Health.

Animals were premedicated with tramadol chloride (4 mg/kg, subcutaneously), and anesthesia was induced and maintained with isoflurane in a non-rebreathing circuit, through a facemask. The animals were positioned in a stereotaxic apparatus, received prophylactic antibiotic with cephalotin (60 mg/kg, subcutaneously) and then, prepared for asseptic surgery. An incision was made in the dorsal midline skin and subcutaneous tissues from T8 to L1, and the muscle and tissue overlying the spinal column were blunt-dissected away revealing the laminae. Using the spiny process of T13 as a landmark, laminectomy of T12 was performed with a pneumatic drill and the lamina was carefully removed to expose the spinal cord. Extradural compression of the spinal cord at the vertebral level of T12 was achieved as previously described [27–30] using a weight of 70 g/cm2. Five minutes later, an intralesional injection was performed according to the experimental protocol. The incision was closed in two layers and the animals were allowed to recover from anesthesia in a warmed (37°C) box.

Post-operative care procedures involved manual expression of the bladder, three times a day, tramadol chloride (2 mg/kg, orally, every eight hours) for three days, and cephalexin (30 mg/kg, orally, twice a day) for five days.

Pharmacological treatment

Five minutes after the incision, 2 μL of treatment was administered into the injury center using a Hamilton microsyringe, as previously described [31]. The animals were distributed into five groups, with six rats each, according to the treatment protocol: placebo treatment with sterile water (PLA), 15 pmol of MVIIC (G15), 30 pmol of MVIIC (G30), 60 pmol of MVIIC (G60), and 120 pmol of MVIIC (G120). Doses were based on studies describing that MVIIC doses of 3 pmol have analgesic effects by blocking the VDCC type P/Q, and those of 100 pmol had side effects [23, 32].

For eight days, experiments focused on clinical observation. All animals were euthanized at Day 8 following SCI. Clinical and histopathological evaluations were carried out by investigators who did not take part into the research.

Clinical evaluation

For three days before SCI, animals were allowed to adapt to the open field arena – that had 90 cm in diameter and three inches high – for 15 minutes to clinical evaluation. After trauma, they were monitored for the presence of widespread tremor, walking in circle or muscle weakness, during the first five hours after the toxin administration and daily until euthanasia [33].

Blood collection

Blood samples were collected prior to experiment and eight days after treatment by caudal vein puncture in two types of tubes, with anticoagulant sodium fluoride in order to access hematologic profile, and without anticoagulant to collect serum and evaluate biochemical profiles, both analyzed immediately.

Hematological parameters

Hematological parameters including red blood cells (RBC), white blood cells (WBC) and hemoglobin concentration (Hb) were determined using Abacus Junior Vet electronic counter (Diatron Messtechnik GmbH, Austria). The RBC indices – namely mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) – were calculated using the RBC count, Hb and hematocrit (Ht) values. Blood smears were prepared on glass slides (26 × 79 mm), fixed with May-Grunwald solution and stained with Giemsa in order to carry out differential counting of leukocytes and total number of platelets [34]. Volume of packed RBC or Ht was determined using a microhematocrit centrifuge (Model Spin 1000®, Micro Spin, USA). The blood was centrifuged to obtain plasma and to determinate total protein by refractometry.

Biochemical parameters

Urea, creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined with the aid of commercial kits from Synermed® (Westfield, USA) and Cobas Mira Classic® chemical analyzer (Global Medical Instrumentations, USA).

Histological analysis of tissue injury

On Day 8 after surgery, the rats were deeply anesthetized with an overdose of sodium thiopental (100 mg/kg), intraperitoneally. The animals were perfused with 300 mL of 0.9% sodium chloride saline followed by 300 mL of 10% phosphate-buffered formalin (pH 7.4). Following perfusion, the brain, heart, liver, kidney, lung, spleen, stomach, lumbar muscle, intestine, pancreas and adrenal were removed and placed overnight in 10% phosphate-buffered formalin. Twenty-four hours later, organ samples were dehydrated in a series of alcohol grades and embedded in paraffin wax. Briefly, 4-μm thick longitudinal sections were stained with routine hematoxylin-eosin (HE) for pathological studies.

Lesion areas were classified in nine grades, according to the histological pattern of intensity (mild, moderate, and severe) and extension (focal, multifocal, and diffuse) of the lesion (Table 1).

Table 1 Bladder scores according to the lesion histological pattern
Full size table

Statistical analysis

All collected data were analyzed using Prism 5® for Windows (GraphPad Software, La Jolla, USA) and were expressed as mean ± standard deviation (SD). Normal distributed data were subjected to analysis of variance (ANOVA), followed by t-paired test between times and Student-Newman-Keuls test between groups. Non-parametric parameters were subjected to Kruskall-Wallis test and Dunn’s post hoc test (p < 0.05).

Results and discussion

In the current experiment, we studied the clinical, hematological, biochemical and tissue histopathological changes to investigate any toxic effects caused by MVIIC. These parameters help to identify possible changes caused by such toxins, the severity of the alterations and which doses can be clinically used in SCI.

Clinical evaluation

Although frequently reported, in the present study no dose provoked muscle weakness or paralysis as seen by Dalmolin et al. [23] at 100 or 300 pmol by intrathecal (IT) route or even shaking at 10 pmol by intracerebroventricular (IC) route. These differences may be attributable to the fact that it was impossible to evaluate variables such as hindlimb locomotor deficit, flaccid paralysis or decreased tail withdrawal response, describe by these authors, because they are similar to clinical signs of SCI. Besides, Malmberg and Yaksh [33] noticed that body shaking occurred 30 minutes after the injection and, at this time, the animal was recovering from anesthesia. Moreover, Dalmolin et al. [23] observed that IC route had more side effects than IT. This fact could be associated to the high density of P/Q-type expressed in nociceptive pathways at the supraspinal site, which emphasizes the powerful effect of MVIIC injected by IC route [35]. It also could be inferred that due to the IL injection into the spinal cord, we did not notice even body shaking as seen at 10 pmol by IC route.

Hematological parameters

In this experiment, an increase in RBC, Ht (Figure 1) and Hb (Figure 2) concentration in all groups may be due to the postoperative stress, restraint and anesthesia prior to euthanasia, leading to a splenic contraction and consequent erythrocyte release [36]. Furthermore, hemoconcentration is most likely a result of the liquid loss due to the weakened condition of rats in the postoperative period and reduced water consumption. Dehydration was not clinically observed, but water intake was reduced as suggested the levels of bottles. Moreover, in the dehydration process, total protein concentration is augmented [37]. Proteins have many functions in the organism and their levels helps in the diagnosis and prognosis of hydration status, inflammation, infection, nutritional status and changes in protein synthesis [38]. In this study, the absolute values of total protein showed no differences among times and groups (Additional file 1), suggesting that hematological changes are likely related to postoperative stress and euthanasia. Nevertheless, these parameters remained close to the reference values for the species [39].

Figure 1
figure 1

Effects of different doses of MVIIC on red blood cells (× 106/μL) and hematocrit. Mean number ± SD of red blood cells and hematocrit of rats submitted to compressive spinal cord injury and treated with placebo (PLA, positive control) or ω-conotoxin MVIIC (G15, 15 pmol MVIIC; G30, 30 pmol MVIIC; G60, 60 pmol MVIIC), preoperatively (white column) and eight days after treatment (gray column) (*p < 0.05; **p < 0.01). Red blood cells and hematocrit normal values are, respectively, 7.62 - 9.99 × 106/μL and 38.5-52%, as described by Giknis and Clifford [39].

Full size image
Figure 2
figure 2

Effects of different doses of MVIIC on hemoglobin (g/dL) and platelets (× 103/μL). Mean number ± SD of hemoglobin and platelets of rats submitted to compressive spinal cord injury and treated with placebo (PLA, positive control) or ω-conotoxin MVIIC (G15, 15 pmol MVIIC; G30, 30 pmol MVIIC; G60, 60 pmol MVIIC), preoperatively (white column) and eight days after treatment (gray column) (*p < 0.05). Hemoglobin and platelets concentration normal values are, respectively, 13.5-17.4 g/dL and 574–1253 × 103/μL, as described by Giknis and Clifford [39].

Full size image

The values of MCV (Additional file 2), MCH (Additional file 3) and MCHC (Additional file 4) did not differ among times or groups, remaining within the physiologic patterns of species.

The increase of platelets (Figure 2) is a common finding in patients who underwent surgical intervention probably by tissue damage and inflammation. The relevance of this event is not clear yet [40–42]. Despite this fact, the parameters were within physiological standards for these animals (574–1253 × 103 cells/μL) [39].

There are no reports of hematologic evaluation in animals that receive MVIIC, and this study shows that it does not cause anemia, hemolysis, vascular changes or interference in the hematologic response when applied by intralesional route.

There was a significant increase of the total leukocyte count in the PLA (7.680 ± 2.140 cells/μL) among the times of collection, not exceeding the maximum limits for the species (Figure 3) (p < 0.05). The leukocytosis can be attributed to stress induced by physical restraint at the time of euthanasia. In acute stress conditions, the release of endogenous glucocorticoids promotes increased blood and lymph circulation so leukocytes pass into the peripheral blood causing physiological leukocytosis [43]. However, only the PLA group showed this significant leukocyte increase. Another possible explanation is that in animals with spinal cord trauma, there is great local inflammatory reaction in PLA group. In contrast, MVIIC groups did not show significant leukocyte augmentation and we can infer a possible anti-inflammatory effect, requiring further investigation.

Figure 3
figure 3

Effects of different doses of MVIIC on total leukocytes (cells/μL) and lymphocytes (cells/μL). Mean number ± SD of total leukocytes and lymphocytes of rats submitted to compressive spinal cord injury and treated with placebo (PLA, positive control) or ω-conotoxin MVIIC (G15, 15 pmol MVIIC; G30, 30 pmol MVIIC; G60, 60 pmol MVIIC), preoperatively (white column) and eight days after treatment (gray column) (*p < 0.05). Total leukocyte and lymphocyte normal values are, respectively, 1980–11060 cells/μL and 1190–9450 cells/μL, as described by Giknis and Clifford [39].

Full size image

There was no change in the absolute number of monocytes (Additional file 5), neutrophils (Additional file 6), and basophils (Additional file 7) among groups or times. Regarding the number of lymphocytes, it can be observed a significant increase (p < 0.05) in animals of PLA group (5.750 ± 1.250) eight days after the injury (Figure 3). Lymphocytes are the major circulating cells of rats, so the leucocytosis is probably due to lymphocytosis. Moreover, there was no difference among times and groups related to the absolute number of eosinophils, showing that MVIIC did not cause sensitization processes indicated by eosinophilia [44].

As seen before, there was no toxic effect of treatment and, therefore, the toxin at the tested doses did not cause any hematological change. These results are unprecedented in the literature.

Biochemical parameters

Biochemical analysis is an important variable for evaluation in studies on toxins which reflects hepatic and renal function. Some enzymes such as AST and ALT are used to evaluate liver function, a key organ for drug metabolism. They reveal abnormalities, and deleterious effects of toxins may increase their levels, especially ALT, which is more specific for liver changes in rats [45]. In our findings, there was no difference in the values of AST and ALT among time points (Figure 4), so there was no toxic effect on liver tissue, which was confirmed by liver histopathology. In addition to reflecting liver abnormalities, AST is used to assess myocardial infarction. Thus, according to our results, it can be stated that there was no significant cardiac muscle damage confirmed by heart histopathology.

Figure 4
figure 4

Effects of different doses of MVIIC on biochemical parameters. Mean number ± SD of alanine aminotransferase (ALT) (U/L), aspartate aminotransferase (AST) (U/L), urea (mg/dL) and creatinine (mg/dL) in different times (preoperatively and eight days after injection) (p < 0.05). Controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). ALT, AST, urea and creatinine normal values are, respectively, 19–48 U/L, 63–175 U/L, 10.7-20 mg/dL and 0.3-0.5 mg/dL, as described by Giknis and Clifford [39].

Full size image

Furthermore, renal excretion is a major route for drug elimination and their commitment may be evidenced by increases in serum creatinine and urea, indicators of glomerular filtration. Creatinine is considered a more reliable indicator that may be affected by the influence of extrarenal factors, and it is the final product of energy used by muscle tissue [46]. Its concentration in blood depends on muscle injury, physical efforts and also meat intake in the case of carnivorous. It is filtered by the renal glomeruli, so renal glomerular lesions are verified by increased creatinine and urea [46, 47]. These values did not differ among groups and times (Figure 4). However, high levels of urea and creatinine in comparison with the physiological data set by Giknis and Clifford [39] were noticed, both preoperatively and on the eighth day after trauma. Therefore, different standard physiological data from the literature were observed.

Histopathological evaluation

Histopathological studies revealed no significant abnormalities in tissue of brain, lung, heart, liver, adrenal, muscle, pancreas, spleen, and stomach in all groups.

In our study kidney tissues showed degenerative changes, especially when treated with 120-pmol doses of MVIIC, including degeneration in the Bowman’s space, glomerular and tubular epithelial cells with deposition of eosinophilic material (amyloid) inside renal tubules, atrophy and glomerular sclerosis [48]. The 120-pmol dose significantly differed from others groups (p < 0.05) (Figures 5 and 6). This type of injury may lead to renal failure and have been observed in animals after spinal trauma following spider or scorpion envenomation [49–51]. It seems that in addition to amyloidosis caused by SCI, the 120-pmol dose intensified the kidney injury, even without increased levels of urea and creatinine, since it occurs when over 75% of renal function is lost [47]. Although there is no record of systemic effects of MVIIC, the highest dose increased renal changes, possibly by a direct glomerulopathy to renal tubule, or indirect, inflammatory response, as seen with others drugs [52].

Figure 5
figure 5

Median scores for renal amyloidosis in rats submitted to spinal cord injury. Controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Lowercase letters express statistically differences among groups, after eight days of spinal cord injury (Kruskal-Wallis test and Dunn’s post hoc test; p < 0.05).

Full size image
Figure 6
figure 6

Light microscopy of renal longitudinal sections of rats stained with hematoxylin-eosin. (A) Kidney showing mild degenerative changes in tubules (arrow) and glomeruli (asterisk) in the control group (PLA) (210×) when compared to (B) the group that received 120 pmol of MVIIC (G120), revealing severe degeneration in the Bowman’s space, glomerular (asterisk) and tubular epithelial cells with deposition of eosinophilic material inside renal tubules (arrow) (207×).

Full size image

Conclusion

Our results suggest that MVIIC at 15, 30 and 60 pmol are safe to be used via intralesional route after spinal cord injury. The 120-pmol dose of MVIIC was detrimental to renal tissue, but it was not enough to change the renal function. More studies are necessary to investigate other routes of MVIIC administration and its possible side effects.

Ethics committee approval

All procedures of the current research were performed in accordance with ethical principles of animal experimentation adopted by the Ethics Committee on Animal Experimentation of the Federal University of Minas Gerais (CETEA/UFMG), which approved the present study under protocol n. 075/10.

Abbreviations

SCI:

Spinal cord injury

MVIIC:

ω-conotoxin MVIIC

CCB:

Calcium channel blockers

VDCC:

Voltage-dependent calcium channels

PLA:

Placebo

G15:

15 pmol of MVIIC

G30:

30 pmol of MVIIC

G60:

60 pmol of MVIIC

G120:

120 pmol of MVIIC

RBC:

Red blood cells

WBC:

White blood cells

Hb:

Hemoglobin

MCV:

Mean corpuscular volume

MCH:

Mean corpuscular hemoglobin

MCHC:

Mean corpuscular hemoglobin concentration

HT:

Hematocrit

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

HE:

Hematoxylin-eosin

SD:

Standard deviation.

References

  1. Tator CH: Strategies for recovery and regeneration after brain and spinal cord injury. Inj Prev 2002, 8(Suppl 4):iv33-iv36.

    PubMed Central  PubMed  Google Scholar 

  2. Rossignol S, Schwab M, Schwartz M, Fehlings MG: Spinal cord injury: time to move? J Neurosci 2007, 27(44):11782–11792. 10.1523/JNEUROSCI.3444-07.2007

    CAS  PubMed  Google Scholar 

  3. Chiu WT, Lin HC, Lam C, Chu SF, Chiang YH, Tsai SH: Epidemiology of traumatic spinal cord injury: comparisons between developed and developing countries. Asia Pac J Public Health 2010, 22(1):9–18. 10.1177/1010539509355470

    PubMed  Google Scholar 

  4. Isaac L, Pejic L: Secondary mechanisms of spinal cord injury. Surg Neurol 1995, 43(5):484–485. 10.1016/0090-3019(95)80094-W

    CAS  PubMed  Google Scholar 

  5. Amar AP, Levy ML: Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury. Neurosurgery 1999, 44(5):1027–1039. 10.1097/00006123-199905000-00052

    CAS  PubMed  Google Scholar 

  6. Lu J, Ashwell KW, Waite P: Advances in secondary spinal cord injury. Spine 2000, 25(14):1859–1866. 10.1097/00007632-200007150-00022

    CAS  PubMed  Google Scholar 

  7. Carlson GD, Gorden C: Current developments in spinal cord injury research. Spine J 2002, 2(2):116–128. 10.1016/S1529-9430(01)00029-8

    PubMed  Google Scholar 

  8. Hall ED, Springer JE: Neuroprotection and acute spinal cord injury: a reappraisal. NeuroRx 2004, 1(1):80–100. 10.1602/neurorx.1.1.80

    PubMed Central  PubMed  Google Scholar 

  9. Liu WM, Wu JY, Li FC, Chen QX: Ion channel blockers and spinal cord injury. J Neurosci Res 2011, 89(6):791–801. 10.1002/jnr.22602

    CAS  PubMed  Google Scholar 

  10. Choi DW: Excitotoxicity cell death. J Neurobiol 1992, 23(9):1261–1276. 10.1002/neu.480230915

    CAS  PubMed  Google Scholar 

  11. Lanz O, Bergman R, Shell L: Initial assessment of patients with spinal cord trauma. Vet Med 2000, 95: 851–853.

    Google Scholar 

  12. Rajendra W, Armugam A, Jeyaseelan K: Neuroprotection and peptide toxins. Brain Res Brain Res Rev 2004, 45(2):125–141. 10.1016/j.brainresrev.2004.04.001

    CAS  PubMed  Google Scholar 

  13. Hillyard DR, Monje VD, Mintz IM, Bean BP, Nadasdi L, Ramachandran J, Miljanich G, Azimi-Zoonooz A, McIntosh JM, Cruz LJ, Imperial JS, Olivera BM: A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron 1992, 9(1):69–77. 10.1016/0896-6273(92)90221-X

    CAS  PubMed  Google Scholar 

  14. Olivera BM, Miljanich G, Ramachandran J, Adams ME: Calcium channel diversity and neurotransmitter release: the omega-conotoxins and omega-agatoxins. Annu Rev Biochem 1994, 63: 823–867. 10.1146/annurev.bi.63.070194.004135

    CAS  PubMed  Google Scholar 

  15. Uchitel OD: Toxins affecting calcium channels in neurons. Toxicon 1997, 35(8):1161–1191. 10.1016/S0041-0101(96)00210-3

    CAS  PubMed  Google Scholar 

  16. Minami K, Raymond C, Martin-Moutot N, Ohtake A, Renterghem CV, Takahashi M, Seagar MJ, Mori Y, Sato K: Role of Thr11 in the binding of ω-conotoxin MVIIC to N-type Ca2+ channels. FEBS Lett 2001, 491(1–2):127–130.

    CAS  PubMed  Google Scholar 

  17. Liu H, Waards M, Scott VES, Gurnett CA, Lennon VA, Campbell KP: Identification of three subunits of the high affinity ω-conotoxin MVIIC-sensitive Ca2+ channel. J Biol Chem 1996, 271: 13804–13810. 10.1074/jbc.271.23.13804

    CAS  PubMed  Google Scholar 

  18. McDonough SI, Boland LM, Mintz IM, Bean BP: Interactions among toxins that inhibit N-type and P-type calcium channels. J Gen Physiol 2002, 119(4):313–328. 10.1085/jgp.20028560

    PubMed Central  CAS  PubMed  Google Scholar 

  19. Pinheiro ACN, Silva AJ, Prado MA, Cordeiro MN, Richardson M, Batista MC, de Castro Junior CJ, Massenssini AR, Guatimosim C, Romano-Silva MA, Kushmerick C, Gomez MV: Phoneutria spider toxins block ischemia-induced glutamate release, neuronal death, and loss of neurotransmission in hippocampus. Hippocampus 2009, 19(11):1123–1129. 10.1002/hipo.20580

    CAS  PubMed  Google Scholar 

  20. Wright CE, Angus JA: Effects of N-, P- and Q-type neuronal calcium channel antagonists on mammalian peripheral neurotransmission. Br J Pharmacol 1996, 119(1):49–56. 10.1111/j.1476-5381.1996.tb15676.x

    PubMed Central  CAS  PubMed  Google Scholar 

  21. Imaizumi T, Kocsis JD, Waxman SG: The role of voltage-gated Ca++ channels in anoxic injury of spinal cord white matter. Brain Res 1999, 817(1–2):84–92.

    CAS  PubMed  Google Scholar 

  22. Bowersox SS, Gadbois T, Singh T, Pettus M, Wang YX, Luther RR: Selective N-type neuronal voltage-sensitive calcium channel blocker, SNX-111, produces spinal antinociception in rat models of acute, persistent, and neuropathic pain. J Pharmacol Exp Ther 1996, 279(3):1243–1249.

    CAS  PubMed  Google Scholar 

  23. Dalmolin GD, Silva CR, Rigo FK, Gomes GM, Cordeiro MN, Richardson M, Silva MA, Prado MA, Gomez MV, Ferreira J: Antinociceptive effect of Brazilian armed spider venom toxin Tx3–3 in animal models of neuropathic pain. Pain 2011, 152(10):2224–2332. 10.1016/j.pain.2011.04.015

    CAS  PubMed  Google Scholar 

  24. Watters MR, Stommel EW: Marine neurotoxins: envenomations and contact toxins. Curr Treat Options Neurol 2004, 6(2):115–123. 10.1007/s11940-004-0021-8

    PubMed  Google Scholar 

  25. Snutch TP: Targeting chronic and neuropathic pain: the N-type calcium channel comes of age. NeuroRx 2005, 2(4):662–670. 10.1602/neurorx.2.4.662

    PubMed Central  PubMed  Google Scholar 

  26. Takahashi Y, Tsuji O, Kumagai G, Hara CM, Okano HJ, Miyawaki A, Toyama Y, Okano H, Nakamura M: Comparative study of methods for administering neural stem/progenitor cells to treat spinal cord injury in mice. Cell Transplant 2011, 20(5):727–739. 10.3727/096368910X536554

    PubMed  Google Scholar 

  27. Silva CMO, Melo EG, Almeida AERF, Gomez MG, Silva CHO, Rachid MA, Verçosa Júnior D, Vieira NT, França SA: Effect of prednisone on acute experimental spinal cord injury in rats. Arq Bras Med Vet Zootec 2008, 60(3):641–650. 10.1590/S0102-09352008000300018

    CAS  Google Scholar 

  28. Torres BBJ, Caldeira FMC, Gomes MG, Serakides R, Viott AM, Bertagnolli AC, Fukushima FB, Oliveira KM, Gomez MV, Melo EG: Effects of dantrolene on apoptosis and immunohistochemical expression of NeuN in the spinal cord after traumatic injury in rats. Int J Exp Pathol 2010, 91(6):530–536. 10.1111/j.1365-2613.2010.00738.x

    PubMed Central  CAS  PubMed  Google Scholar 

  29. Torres BBJ, Silva CMO, Almeida ÁERF, Caldeira FMC, Gomes MG, Alves EGL, Silva SJ, Melo EG: Experimental model of acute spinal cord injury produced by modified steriotaxic equipment. Arq Bras Med Vet Zootec 2010, 62(1):92–99. 10.1590/S0102-09352010000100013

    Google Scholar 

  30. Torres BBJ, Serakides R, Caldeira F, Gomes M, Melo E: The ameliorating effect of dantrolene on the morphology of urinary bladder in spinal cord injured rats. Pathol Res Pract 2011, 207(12):775–779. 10.1016/j.prp.2011.10.004

    CAS  PubMed  Google Scholar 

  31. Kamada T, Koda M, Dezawa M, Anahara R, Toyama Y, Yoshinaga K, Hashimoto M, Koshizuta S, Nishio Y, Mannoji C, Okawa A, Yamazaki M: Transplantation of human bone marrow stromal cell-derived Schwann cells reduces cystic cavity and promotes functional recovery after contusion injury of adult rat spinal cord. Neuropathology 2011, 31(1):48–58. 10.1111/j.1440-1789.2010.01130.x

    PubMed  Google Scholar 

  32. Silva JF: Avaliação da atividade antinociceptiva espinhal da toxina Tx3–4 do veneno da aranha Phoneutria nigriventer em camundongos. Belo Horizonte: UFMG; 2009.

    Google Scholar 

  33. Malmberg AB, Yaksh TL: Voltage-sensitive calcium channels in spinal nociceptive processing: blockade of N- and P-Type channels inhibits formalin-induced nociception. J Neurosci 1994, 14(8):4882–4890.

    CAS  PubMed  Google Scholar 

  34. Thrall MA, Weiser G, Allison R, Campbell T: Veterinary Hematology and Clinical Chemistry. Baltimore: Lippincott Williams & Wilkins; 2004.

    Google Scholar 

  35. Hillman D, Chen S, Aung TT, Cherksey B, Sugimori M, Llinás RR: Localization of P-type calcium channels in the central nervous system. Proc Natl Acad Sci U S A 1991, 88(16):7076–7080. 10.1073/pnas.88.16.7076

    PubMed Central  CAS  PubMed  Google Scholar 

  36. Weiss DJ, Wardrop JK: Schalm’s Veterinary Hematology. Iowa: Blackwell Publishing; 2010.

    Google Scholar 

  37. Watson ADJ: Erythrocytosis and polycythemia. In Schalm’s Veterinary Hematology. 5th edition. Edited by: Feldman BF, Zinkl JG, Jain NC. Philadelphia: Lippincott Williams & Wilkins; 2000:200–204.

    Google Scholar 

  38. Santana AM, Fagliari JJ, Camargo CMS, Santana AE, Duarte JMB, Silva PRL: Serum protein concentrations of captive brown brocket deer ( Mazama gouazoubira ) determined by means of agarosis and sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gel electrophoresis. Arq Bras Med Vet Zootec 2008, 60(6):1560–1563. 10.1590/S0102-09352008000600039

    CAS  Google Scholar 

  39. Giknis MLA, Clifford CB: Clinical Laboratory Parameters for crl:WI (han). Massachusetts: Charles River Lab; 2008:1–17. http://www.criver.com/files/pdfs/rms/wistarhan/rm_rm_r_wistar_han_clin_lab_parameters_08.aspx

    Google Scholar 

  40. Bunting RW, Doppelt SH, Lavine LS: Extreme thrombocytosis after orthopaedic surgery. J Bone Joint Surg 1991, 73-B: 687–688.

    Google Scholar 

  41. Valade N, Decailliot F, Rébufat Y, Heurtematte Y, Duvaldestin P, Stéphan F: Thrombocytosis after trauma: incidence, aetiology, and clinical significance. Br J Anaesth 2005, 94(1):18–23.

    CAS  PubMed  Google Scholar 

  42. Salim A, Hadjizacharia P, DuBose J, Kobayashi L, Inaba K, Chan LS, Marqulies DR: What is the significance of thrombocytosis in patients with trauma? J Trauma 2009, 66(5):1349–1354. 10.1097/TA.0b013e318191b8af

    PubMed  Google Scholar 

  43. Fukui Y, Sudo N, Yu XN, Nukina H, Sogawa H, Kubo C: The restraint stress-induced reduction in lymphocyte cell number in lymphoid organs correlates with the suppression of in vivo antibody production. J Neuroimmunol 1997, 79(2):211–215. 10.1016/S0165-5728(97)00126-4

    CAS  PubMed  Google Scholar 

  44. Jain NC: Essentials of Veterinary Hematology. Philadelphia: Lea & Febiger; 1993.

    Google Scholar 

  45. Tennant BC, Center SA: Hepatic function. In Clinical Biochemistry of Domestic Animals. 6th edition. Edited by: Kaneko JJ, Harvey JW, Bruss ML. San Diego: Academic Press; 2008:379–412.

    Google Scholar 

  46. Magro MCS, Vattimo MFF: Evaluation of renal function: creatinine and other biomarkers. Rev Bras Ter Int 2007, 19(2):182–185. 10.1590/S0103-507X2007000200007

    Google Scholar 

  47. Braun JP, Lefebvre HP: Kidney function and damage. In Clinical Biochemistry of Domestic Animals. 6th edition. Edited by: Kaneko JJ, Harvey JW, Bruss ML. San Diego: Academic Press; 2008:485–528.

    Google Scholar 

  48. Di Bartola SP, Tarr MJ, Webb DM, Giger U: Familial renal amyloidosis in Chinese Shar-Pei dogs. J Am Vet Med Assoc 1990, 197(4):483–487.

    CAS  Google Scholar 

  49. Wall BM, Huch KM, Mangold TA, Steere EL, Cooke R: Risk factors for development of proteinuria in chronic spinal cord injury. Am J Kidney Dis 1999, 33(5):899–903. 10.1016/S0272-6386(99)70423-3

    CAS  PubMed  Google Scholar 

  50. Tambourgi DV, Petricevich VL, Magnoli FC, Assaf SL, Jancar S, Dias Da Silva W: Endotoxemic-like shock induced by Loxosceles spider venoms: pathological changes and putative cytokine mediators. Toxicon 1998, 36(2):391–403. 10.1016/S0041-0101(97)00063-9

    CAS  PubMed  Google Scholar 

  51. Dehghani R, Khamechian T, Rafiezadeh MTZ, Nasajizavareh M: Histopathological assessment of Hemiscorpius lepturus venom effects on rats. Iran J Leg Med 2004, 10: 202–206.

    Google Scholar 

  52. Hill GS: Drug-associated glomerulopathies. Toxic Pathol 1986, 14(1):37–44. 10.1177/019262338601400105

    CAS  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge the support from the State of Minas Gerais Research Foundation (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq) for supporting the project, the Coordination for the Improvement of Higher Education Personnel (CAPES) for granting a scholarship and Cristália Lab for providing the medications. All these organizations did not have access to the research and had no role in data collection, analysis, interpretation, writing or decision to publish. None of the authors was paid.

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Authors and Affiliations

  1. Departamento de Clínica e Cirurgia Veterinária, Escola de Veterinária, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha, Belo Horizonte, MG CEP 30123-970, Brasil

    Karen M Oliveira, Carla Maria O Silva, Isabel R Rosado, Fabíola B Fukushima, Luiza Anna FV Assumpção, Saira MN Neves, Guilherme R Motta, Fernanda F Garcia, Marília M Melo & Eliane G Melo

  2. Departament of Agrarian and Environmental Sciences, State University of Santa Cruz, Ilhéus, Bahia State, Brazil

    Mário Sérgio L Lavor

  3. National Institute of Sciences and Technology on Molecular Medicine, School of Medicine, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais State, Brazil

    Marcus Vinícius Gomez

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  1. Karen M Oliveira
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  2. Carla Maria O Silva
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Corresponding author

Correspondence to Karen M Oliveira.

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Competing interests

The authors declare that there are no competing interests.

Authors’ contributions

KMO was the main responsible, participated in all stages of the experiment that was part of her master's project. CMOS contributed to the all surgeries and care for animals. MSLL participated in the study design, contributed to the surgeries and helped to draft the manuscript. IRR contributed to some surgeries and care of animals. FBF participated in the study design, contributed to the surgeries and helped to draft the manuscript. ALVFA took care of the animals, assisting with medications, bladder massage and clinical evaluation. SMNN assisted with the microscopic evaluation of tissue. GRM took care of the animals, assisting with medications, bladder massage and clinical evaluation. FFG helped with hematological examinations. MVG participated in the study design and coordination of the project. MMM participated in the study design and helped to draft the manuscript. EGM and MVG participated in the study design, coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Electronic supplementary material

40409_2013_39_MOESM1_ESM.tiff

Additional file 1:Effects of different doses of MVIIC on total protein levels. After spinal cord injury, controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Values represent the means ± SD of six animals at each time. (TIFF 782 KB)

40409_2013_39_MOESM2_ESM.tiff

Additional file 2:Effects of different doses of MVIIC on median corpuscular volume. After spinal cord injury, controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Values represent the means ± SD of six animals at each time. (TIFF 530 KB)

40409_2013_39_MOESM3_ESM.tiff

Additional file 3:Effects of different doses of MVIIC on median corpuscular hemoglobin. After spinal cord injury, controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Values represent the means ± SD of six animals at each time. (TIFF 449 KB)

40409_2013_39_MOESM4_ESM.tiff

Additional file 4:Effects of different doses of MVIIC on mean corpuscular hemoglobin concentration. After spinal cord injury, controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Values represent the means ± SD of six animals at each time. (TIFF 649 KB)

40409_2013_39_MOESM5_ESM.tiff

Additional file 5:Effects of different doses of MVIIC on monocyte levels. After spinal cord injury, controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Values represent the means ± SD of six animals at each time. (TIFF 560 KB)

40409_2013_39_MOESM6_ESM.tiff

Additional file 6:Effects of different doses of MVIIC on neutrophil levels. After spinal cord injury, controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Values represent the means ± SD of six animals at each time. (TIFF 544 KB)

40409_2013_39_MOESM7_ESM.tiff

Additional file 7:Effects of different doses of MVIIC on basophil levels. After spinal cord injury, controls were injected with sterile water (PLA) and other groups received different doses of MVIIC (15, 30, 60 and 120 pmol). Values represent the means ± SD of six animals at each time. (TIFF 266 KB)

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Oliveira, K.M., Silva, C.M.O., Lavor, M.S.L. et al. Systemic effects induced by intralesional injection of ω-conotoxin MVIIC after spinal cord injury in rats. J Venom Anim Toxins Incl Trop Dis 20, 15 (2014). https://doi.org/10.1186/1678-9199-20-15

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Keywords

Journal of Venomous Animals and Toxins including Tropical Diseases

ISSN: 1678-9199

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