Electrical stimulation shifts healing/scarring towards regeneration in a rat limb amputation model.

Different species respond differently to severe injury, such as limb loss. In species that regenerate, limb loss is met with complete restoration of the limbs' form and function, whereas in mammals the amputated limb's stump heals and scars. In in vitro studies, electrical stimulation (EStim) has been shown to promote cell migration, and osteo- and chondrogenesis. In in vivo studies, after limb amputation, EStim causes significant new bone, cartilage and vessel growth. Here, in a rat model, the stumps of amputated rat limbs were exposed to EStim, and we measured extracellular matrix (ECM) deposition, macrophage distribution, cell proliferation and gene expression changes at early (3 and 7 days) and later stages (28 days). We found that EStim caused differences in ECM deposition, with less condensed collagen fibrils, and modified macrophage response by changing M1 to M2 macrophage ratio. The number of proliferating cells was increased in EStim treated stumps 7 days after amputation, and transcriptome data strongly supported our histological findings, with activated gene pathways known to play key roles in embryonic development and regeneration. In conclusion, our findings support the hypothesis that EStim shifts injury response from healing/scarring towards regeneration. A better understanding of if and how EStim controls these changes, could lead to strategies that replace scarring with regeneration.

Multi-channel orbicularis oculi stimulation to restore eye-blink function in facial paralysis.

Facial paralysis due to facial nerve injury results in the loss of function of the muscles of the hemiface. The most serious complication in extreme cases is the loss of vision. In this study, we compared the effectiveness of single- and multiple-channel electrical stimulation to restore a complete and cosmetically acceptable eye blink. We established bilateral orbicularis oculi muscle (OOM) paralysis in eight dogs; the OOM of one side was directly stimulated using single-channel electrical stimulation and the opposite side was stimulated using multi-channel electrical stimulation. The changes in the palpebral fissure and complete palpebral closure were measured. The difference in current intensities between the multi-channel and single-channel simulation groups was significant, while only multi-channel stimulation produced complete eyelid closure. The latest electronic stimulation circuitry with high-quality implantable electrodes will make it possible to regulate precisely OOM contractions and thus generate complete and cosmetically acceptable eye-blink motion in patients with facial paralysis.

Electrically stimulated detrusor myoplasty.

PURPOSE: Many children with spina bifida and other causes of neurogenic bladder rely on clean intermittent catheterization to empty the hyporeflexic or areflexic bladder. Direct bladder and sacral nerve root stimulation have been met with limited success. We studied the electrical stimulation of a rectus abdominis muscle flap wrapped around the bladder to achieve bladder contractility and emptying. MATERIALS AND METHODS: The feasibility of performing rectus detrusor myoplasty in humans was first studied in 8 cadavers. In male and female cadavers it was possible to wrap the distended bladder completely with the rectus abdominis muscle. The rectus abdominis muscle was surgically dissected with preservation of its insertion on the pubis bone and rotation of its mid section behind the bladder to effect a complete bladder wrap. The deep inferior epigastric artery and veins, and 2 most caudal intercostal nerves were preserved. This unilateral rectus abdominis muscle flap was then electrically stimulated with 2 pairs of bipolar electrodes inserted into the muscle near the nerve entrance. Stimulation frequencies of 40, 60 and 80 Hz. were used in each of the 8 dogs. The increase in intravesical pressure over baseline, compliance and post-void residual were measured. Paired Student's t tests were used for statistical comparisons. RESULTS: The increase in intravesical pressure ranged 35 +/- 5 to 45 +/- 7 cm. H2O at stimulation frequency 40 and 80 Hz., respectively. Post-void residual was 27 +/- 4%, 22 +/- 3% and 26 +/- 3% at stimulation frequencies 40, 60 and 80 Hz., respectively. Intravesical pressure was significantly increased over baseline bladder pressure (p <0.05). CONCLUSIONS: Electrically stimulated detrusor myoplasty results in uniform increases in intravesical pressure and reasonable bladder emptying in an animal model. We are currently investigating detrusor myoplasty in a chronic study to determine whether it can be used for enhanced bladder emptying in children with poor detrusor contractility.

Topical application of cod liver oil ointment accelerates wound healing: an experimental study in wounds in the ears of hairless mice.

The effect of topical cod liver oil ointment on the rate of wound epithelialisation and neovascularisation was studied using the hairless mouse ear wound model (experiment I). The effect of local application of vitamin A in increasing concentrations was tested in the same model (experiment II). Experiment I: Bilateral standardised full thickness dermal wounds were created on the ears of 42 mice divided into three groups: group I: 25% cod liver oil ointment (n = 10) applied topically to one ear and vehicle (vaseline) to the other; group II: 25% cod liver oil ointment (n = 10) and saline; and group III: vehicle (n = 22) and saline. Experiment II: Using the same model and procedures wounds were made on 12 mice randomised to vitamin A treatment in various doses (250-3000 IU/g) on one ear and vehicle (vaseline) on the other ear. Using in-vivo microscopy and digitised planimetry, wound epithelialisation and neovascularisation were measured at regular intervals until the processes were complete. Wounds treated with 25% cod liver oil ointment epithelialized significantly (p < 0.05) faster (mean (SEM) 8.9 (0.7) days) than control ears treated with vehicle alone (13.9 (1.9) days). Neovascularisation developed significantly faster (p < 0.01) in the ears treated with cod liver oil ointment (22.5 (1.3) days) compared with their vehicle control (29.1 (0.6) days). Neovascularisation was also significantly (p < 0.05) faster in the ears treated with cod liver oil ointment (23.1 (1.4) days) than in those treated with saline (26.8 (1.1) days). There was no significant difference in speed of epithelialisation between cod liver oil ointment and saline. The vitamin A dose study showed that epithelialisation and neovascularisation of the vitamin A treated wounds pass at the same rate as wounds treated with cod liver oil ointment. In conclusion, topical 25% cod liver oil ointment significantly accelerated both the epithelial and the vascular component of healing compared with saline. Vitamin A seems to have an important role in accelerating wound healing and could be the active component in cod liver oil.

Vascular delay and administration of basic fibroblast growth factor augment latissimus dorsi muscle flap perfusion and function.

Ischemia of the distal latissimus dorsi muscle flap occurs when the entire muscle is acutely elevated. Although this level of ischemia may not be critical if the muscle is to be used as a conventional muscle flap, the ischemia causes decreased distal muscle function if it is used for dynamic muscle flap transfer. This experiment was designed to determine whether or not the administration of exogenous basic fibroblast growth factor (bFGF), combined with a sublethal ischemic insult (i.e., vascular delay), would further augment muscle perfusion and function. Both latissimus dorsi muscles of nine canines were subjected to a bipedicle vascular delay procedure immediately followed by thoracodorsal intraarterial injection of 100 microg of bFGF on one side and by intraarterial injection of vehicle on the other. Ten days later, both latissimus dorsi muscles were raised as thoracodorsally based island flaps, with perfusion determined by laser-Doppler fluximetry. The muscles were wrapped around silicone chambers, simulating cardiomyoplasty, and stimulating electrodes were placed around each thoracodorsal nerve. The muscles were then subjected to an experimental protocol to determine muscle contractile function. At the end of the experiment, latissimus dorsi muscle biopsies were obtained for measurement of bFGF expression. The results demonstrated that the administration of 100 microg of bFGF immediately after the vascular delay procedure increases expression of native bFGF. In the distal and middle muscle segments, it also significantly increased muscle perfusion by approximately 20 percent and fatigue resistance by approximately 300 percent. The administration of growth factors may serve as an important adjuvant to surgical procedures using dynamic muscle flap transfers.

Electrically stimulated free-flap graciloplasty for urinary sphincter reconstruction: a new surgical procedure.

In electrically stimulated (dynamic) graciloplasty for urinary incontinence, the gracilis muscle is transposed into the pelvis, and the distal part is used to reconstruct a neosphincter. Clinical outcomes using this technique have been disappointing due to stricture of the urethra caused by ischemia in the distal part of the gracilis and limited gracilis length available for neosphincter construction. Furthermore, the urethra is twisted by the contracting gracilis, rather than circumferentially squeezed. The purpose of the present study was to test the anatomical and functional feasibility of a new surgical approach to reconstruct a urinary sphincter, using the gracilis muscle as a free flap. In 12 human cadavers, the anatomical feasibility for creating a neosphincter by using the gracilis free flap was determined. In all cases, transfer of the gracilis muscle into the pelvis as a free flap (with the nerve intact) was feasible, and ample muscle was available to construct a neosphincter around the bladder neck. Gracilis neosphincter function was studied in seven dogs. The left gracilis muscle was subjected to transfer into the pelvis as an innervated free flap to create a neosphincter around the urethra. The right (control) gracilis muscle was lifted as a single pedicle flap, remained in situ, and was wrapped around a stent to mimic the urethra. Function (expressed as peak pressure generation and fatigue rate) and surface perfusion were determined for all gracilis muscles. In each dog, both sides were compared using the paired Student's t test for statistical analysis, and no significant difference was measured for the two groups. In conclusion, an innervated gracilis free flap can be used to create a neosphincter around the bladder neck. In an acute study in dogs, function and perfusion of the innervated gracilis free flap are not compromised.

Platelet-derived thromboxane A2 decreases microvascular perfusion after arterial repair.

Previous work suggests that cod liver oil helps to protect the microcirculation from the consequence of thromboembolic events. The possibility that altered synthesis of thromboxane A2 accounts for the protective effects seen with cod liver oil was investigated in the present study. This was done using the combined thromboxane A2 synthetase inhibitor and thromboxane A2-prostaglandin H2 receptor blocker R68070 (Ridogrel). A standardized microvascular injury was inflicted on the right iliac artery of the rat to generate emboli. The downstream cremaster muscle was used to visualize the passage of the ensuing emboli and to assess the effects of this arterial injury on capillary perfusion and arteriole diameters. The number of visible emboli was not changed by either cod liver oil diet or Ridogrel administration. However, capillary perfusion was preserved by using cod liver oil (n = 7) and was significantly increased by using Ridogrel (n = 7) in comparison with untreated controls (n = 7) in which capillary perfusion was decreased because of the emboli. The administration of Ridogrel to cod liver oil-treated animals (n = 7) provided no additive benefit. The percentage change in A-2 vessel diameters in cod liver oil-treated (n = 7) animals was no different from the control group (n = 7). Ridogrel (n = 7), on the other hand, produced a significant increase in A-3 vessel (n = 21) diameters, but its effects were comparatively less in the cod liver oil-treated animals (n = 7). The formation of platelet aggregates (emboli) appears relatively independent of thromboxane A2 in the rat. Ridogrel is very effective in protecting the microcirculation, and these effects appear to be mediated by A-3 vasodilatation, which, therefore, is at least partially thromboxane A2-dependent. The positive effects of cod liver oil may be mediated by a mechanism that reduces thromboxane A2 synthesis, but further studies are necessary.

The effects of heparin and dietary fish oil on embolic events and the microcirculation downstream from a small-artery repair.

In recent studies we demonstrated that platelet emboli induced by arterial injury impair the microcirculation. The present study was performed to determine if heparin or dietary cod liver oil reduces the incidence of emboli following the same arterial injury and, in turn, whether these agents prevent the associated decrease in microcirculatory blood flow. The cremaster muscle of 29 male Sprague-Dawley rats was isolated on a single neurovascular bundle consisting of the iliac artery and vein and the genitofemoral nerve. Emboli were generated by a thrombogenic injury of the iliac artery, and their number and their subsequent effect on capillary perfusion in the downstream microcirculation were measured. Nine animals received heparin (10-unit IV bolus plus 10 units/hour IV infusion), 10 were fed cod liver oil (10 percent by weight of food) for 3 weeks prior to the experiment, and 9 animals receiving no treatment served as controls. The number of emboli was significantly reduced in the heparin group, but there was no accompanying improvement in capillary perfusion. In contrast, in the cod liver oil group, the number of emboli was not reduced, but there was significant improvement in capillary perfusion. These findings suggest that the harmful effect that platelet emboli have on the microcirculation is probably biochemical in nature (vasoconstriction) rather than related to simple mechanical obstruction to flow.

Reduced neutrophil superoxide generation and plasma lipoperoxide levels in pigs fed with cod liver oil.

Circulating neutrophils, isolated from pigs fed for 8 weeks with a diet supplemented with CLO, had an accentuated n-3 fatty acid incorporation into the plasma membrane, as evidenced by an approximately four-fold greater n-3/n-6 ratio as compared with the control diet group. Moreover, the neutrophils of the CLO fed pigs produced less superoxide when stimulated with PMA or f-MLP, as well as showing a more prolonged latency period before O2(-)-generation. In the plasma of pigs fed with CLO there were higher levels of thiobarbituric reactive material and lipofuscin, while the content of GSSG was similar in both dietary groups. The results of this study indicate that dietary supplementation with CLO reduces the activation of circulating neutrophils and favours the presence in the plasma of lipoperoxides.