Interact CardioVasc Thorac Surg 2009;9:411-415. doi:10.1510/icvts.2009.202150
© 2009 European Association of Cardio-Thoracic Surgery
Institutional report - Experimental |
Experimental selective sympathicotomy (ramicotomy) and sympathetic regeneration
Humberto Alves Oliveiraa,*,
Manoel Ximenes, IIIa,
Flávio Brito Filhoa,
Paulo H.C. Carvalhob,
José Belarmino GamaFilhob,
Edwin Roger Parrac,
Vera Luiza Capelozzic and
José Ribas Milanez DeCamposd
a Thoracic Surgery Unit, Hospital de Base of Federal District, Brasília, DF, Brazil
b Center for the Study and Technology in Health Sciences, CETREX, Brasília, DF, Brazil
c Department of Pathology, School of Medicine, University of São Paulo, SP, Brazil
d Department of Cardio-Thoracic Surgery, Heart Institute, University of São Paulo, SP, Brazil
Received 8 January 2009;
received in revised form 9 June 2009;
accepted 12 June 2009
 Presented at 13th Congress of the Belgian Association for Cardiothoracic Surgery (BACTS), Brussels, November 15, 2008.
*Corresponding author. AOS 7 Bloco C Apart 509, Octogonal, Zip code: 70.660-073, Brasília, DF, Brazil. Tel.: +55-61-9968-5003; fax: +55-61-3577-4225.
E-mail address: humberto.tor{at}ig.com.br (H.A. Oliveira).
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Abstract
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Ramicotomy is a surgical procedure, with less adverse effects than conventional sympathectomy, however, it was abandoned due to the high recurrence rate. Twenty-eight pigs underwent bilateral videothoracoscopic ramicotomy and were divided into five groups. The animals were sacrificed at 15th, 45th, 90th, 135th and 180th postoperative days (POD). The segments were removed and evaluated for macroscopic regeneration and histological analysis. The data were compared to the control group of 10 intact segments of the sympathetic. There was no macroscopic regeneration on the 15th POD, and present on 41.6% on the 180th POD (P<0.05). The Schwann cells presented a similar evolution in both rami beginning at the 45th POD, with a smaller count in the gray rami. The collagen and reticular fibers presented a negative correlation (r=–0.414; P<0.01). The deposition of the collagen fibers was greater in the gray rami with a peak deposition on the 135th POD and a diminishing rate in the 180th POD (P<0.05). Ramicotomy allows complete section of all rami communicantes of the sympathetic ganglia. The histological regeneration might be greater than the recurrence rates of clinical symptoms seen in a human being due to non-functional regenerations.
Key Words: Sympathectomy; Video-assisted thoracic surgery; Wallerian degeneration; Schwann cells; Ramicotomy
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1. Introduction
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Among the unwanted side effects following thoracic sympathectomy for primary palmar and axillary hyperhidrosis, reflex sweat is the most important [1, 2]. With the purpose of using a less aggressive surgical approach and lesser imbalance of the autonomic nervous system, the ramicotomy was proposed [3]. This procedure carries a high recurrence rate (20%) mainly due to the incomplete section of all rami communicantes in the operated ganglia, as well as the development of new conducting pathways [4, 5]. The aim of this investigation was to analyze morphologically the rami communicantes in different stages after experimental ramicotomy in pigs and to study temporal aspects as well as histological indicators of the cicatrization process and neural regeneration.
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2. Materials and methods
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The twenty-eight swine received antibiotic prophylaxis and analgesic medication. Bilateral selective endoscopic sympathicotomy was performed under general anesthesia. They were randomized and sacrificed within 15 postoperative days (POD) and every 45 POD until six months after the procedure as follows:- Group-I (n=4): 15th POD.
- Group-II (n=6): 45th POD.
- Group-III (n=6): 90th POD.
- Group-IV (n=6): 135th POD.
- Group-V (n=6): 180th POD.
Ten animals with intact ganglia made the control group (Group-0). The procedure initiated identifying the sympathetic trunk over the 3th and 4th ribs which was elevated with a hook and sectioning all structures connected to the trunk, allowing severing of all rami communicantes to the ganglia (Fig. 1).
The animals were sacrificed and submitted to a thoracolaparotomy for resection of segments of the sympathetic trunk when macroscopic regeneration was evaluated (Fig. 2).
The material was cut into sections and stained:- Hematoxylin and Eosin: identification of the sympathetic trunk, ganglia and rami communicantes allowing a semi-quantitative analysis reported as degree of inflammation (Fig. 3a);
- Picrosirius: quantification of the neural area filled with thick and thin collagen fibers, according to the tones of birefringence when analyzed through polarized light (Fig. 3b);
- Weil: differentiation between the rami communicantes rich in myelin, in dark gray tones and the myelin poor rami, in light gray tones (Fig. 3c);
- Reticulin Gomori: the total area of the rami communicantes occupied by reticular fibers was measured by the recognition of light frequencies correspondent to the violet fibers of the sample (Fig. 3d);
- Immunohistochemistry: identification of Schwann cells in the nervous fibers through the expression of the S-100 antigen (Fig. 3e). With point counting technique, the Schwann cell density (Ps-100) was quantified [6].
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Fig. 3. Histological sections to demonstrate: (a) HE with moderate inflammation; (b) Picrosirius over polarized light showing collagen fibers; (c) Weil identifying myelinic of amyelinic fibers; (d) Reticulin Gomori identifying reticular fibers; (e) Immunohistochemistry for the Schwann cells.
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2.1. Statistical analysis
The frequency distribution of the variables was defined by the normality test of Shapiro–Wilk. Comparison between the groups was made through the variance analysis with a factor for the numeric variables and the  2 for the categorical variables. To compare the white and gray rami communicantes in the same group and the same variable between both groups, the independent t-test was applied to the numeric variables.
The degree of correlation between variables with normal distribution was evaluated by the Pearson coefficient. In the determination of the P-values, a two-tail distribution was applied to the numeric variables and a one-tail for the categorical ones. The confidence interval was 95%, with significance level  =0.05.
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3. Results
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3.1. Macroscopic regeneration
Due to technical difficulties the number of ramicotomies fell to 51. The frequency distribution of macroscopic regeneration (Table 1) showed a discreet increase, but only the 180th POD had statistical significance.
3.2. Collagen fibers
There was a similar increase in the percentage area of the white and gray rami communicantes filled with collagen fibers, however, the increase was sudden and significant after 135 POD when compared with the control group (Fig. 4).
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Fig. 4. Comparison between the total collagen fibers in the white and gray rami communicantes in each group. Values expressed in mean±S.E.
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Collagen fibers were analyzed separately, observing the thick and thin fibers. After 45 POD the white rami had a significant increase in the thick collagen fibers as compared with the gray rami. Inversely, after 135 POD, the gray rami presented an increasing number of thick collagen fibers when compared with the white rami.
Thin collagen fibers, inversely to the thick ones, presented a greater intensity of deposition in the gray rami communicantes after 45 POD after ramicotomy than white rami.
3.3. Reticular fibers
Fig. 5 shows a significant decrease of reticular fibers until 45 POD when compared to the control group. After 90 POD there was a new deposition of reticular fibers with a greater tendency of deposition in the white rami communicantes.
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Fig. 5. Comparison between reticular fibers in white and gray rami communicantes. Values expressed in mean±S.E.
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3.4. Collagen X reticular fibers
When comparing reticular fibers deposition with the total of collagen fibers in white and gray rami communicantes, it was verified a significant difference in all groups (P<0.01), associated to a moderate negative linear correlation (r=–0.408 and r=–0.414; respectively), demonstrating that the greater the deposition of reticular fibers, the smaller deposition of collagen fibers and consequently, less cicatrization.
3.5. Schwann cells
Fig. 6 demonstrates that ramicotomy had distinctive effects in the number of Schwann cells in rami communicantes, with tendencies of increase in white and decrease in gray rami communicantes.
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Fig. 6. Comparison between the Schwann cells in white and gray rami communicantes. Values expressed in mean±S.E.
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3.6. Inflammatory reaction
The inflammatory response was intense on the 15th POD, with a progressive and significant decrease in all groups, (P<0.01; Table 2).
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4. Discussion
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Several investigators demonstrated the restoration of peripheral nervous system, motor and autonomous by healing or regeneration process and also identified parameters that favor neural regeneration such as proximity of the sectioned axonal stumps and time spent to their approximation after the injury [7, 8].
Our experimental model reproduces the surgical procedure in humans and allowed observation of the entire spectrum of healing and regenerative process of the rami communicantes. The evaluation of sympathetic regeneration during the procedure of re-sympathectomy in humans is described in a very subjective way, attributing the recurrence to incomplete resection, without a histological proof [9]. In this study, there was no macroscopic regeneration on the 15th POD, but in 30% of the cases on the 45th POD, until reaching 41.6% on the 180th POD (P<0.05). The increase of macroscopic regeneration occurred parallel to a progressive decrease of inflammation in these groups, suggesting a stabilization of degenerative and regenerative processes starting on the 135th POD. In the proximity of the section and in the axonal stump, the degenerative process may prolong through several weeks before any signs of regeneration [8]. This could explain the lack of regeneration on the 15th POD, due to the high inflammatory reaction found in this group (Table 2) suggesting an incoming Wallerian degenerative process.
In this experimental model, macroscopic regeneration in the range of 30–40% does not reflect the recurrent symptoms after ramicotomy in humans (20%) [1]. This difference could be explained by: (i) Anatomical regeneration does not imply in functional regeneration, because for that to occur it is necessary an appropriate reconnection of the neural cell [7]; (ii) Regeneration can occur in only one of the rami communicantes, depending on the operated ganglia for palmar of axillary hyperhidrosis, a rami cannot be sufficient for the recurrence of the symptoms; (iii) The evaluation of macroscopic regeneration is subjective and can generate interpretation errors, like mistaking a fibrous rami for a regenerated one [10].
In this study, the sections of the rami communicantes were made at the same level, close to the sympathetic ganglia. So, the preganglionic rami should correspond to the post section segments in the histological evaluation, thus, suffer from complete degeneration. The postganglionic rami correspond to the pre section segments, where the degeneration evolution is interrupted in the first Ranvier nodule closer to the injury [7].
The first parameter related to cicatrization was the deposition of collagen fibers, which presented a peak on the 135th POD, with an important decrease on the 180th POD, this could be attributed to the cicatrization remodeling, characteristic of the chronic phase of tissue repair [11]. On the 135th and 180th POD, the amount of collagen fibers in the gray rami communicantes was more significant than in the white rami, suggesting a greater cicatrization in the gray rami.
Type I collagen is abundant in the interstice in most tissues and responsible for the cicatrization process. Although, it is not possible to define exactly the type of collagen through Picrosirius techniques, it is known that type I collagen presents as thick fibers under polarized light, so the increasing concentration of these fibers in the gray rami communicantes demonstrates a significant healing process [12].
Thick fibers appeared in decreasing numbers in the postganglionic rami during the first trimester and, inversely, reached higher levels in preganglionic rami during the second trimester (135th POD and 180th POD). The thin fibers appear in elevated levels in postganglionic rami in the first trimester, showing no significant difference in the second trimester.
Reticular fibers, made mainly by type III collagen and with 6–12% sugars, constitute the most relevant conjunctive cover of endoneurum [13] and its maintenance or restoration, after the section of the nerve, should be related to its regeneration. In this study, the reticular fibers diminished after ramicotomy until the 45th POD, suggesting absorption of part of the endoneurum in the initial period of Wallerian degeneration. After that, reticular fibers showed a continuous and minor elevation until the 180th POD, coinciding with the findings of macroscopic regeneration. This elevation of reticular fibers on the 45th POD and 180th POD was less evident in the gray rami communicantes, suggesting a minor level of regeneration.
When comparing the collagen and reticular fibers, we found a negative correlation (r=–0.408 and r=–0.414, respectively; P<0.01). The collagen fiber is the main component in the cicatrization process [12], and the reticular fibers form a delicate net of fibers under the basal membrane of the Schwann cells, providing a cellular remodeling arrangement preserving a space for extra cellular molecular fluid movement of the endoneurum [14]. Considering their distinct functions, the negative correlation between them suggests that the rami that have the most reticular fibers and less collagen would have greater chances of regeneration.
The Schwann cells play an active role in the Wallerian degeneration, helping the macrophages to reabsorb myelin and essential in the regeneration of the axonal distal stump, multiplying and becoming the Büngner bands [15]. In the control group, the amount of Schwann cells in the rami communicantes was similar in the fibers rich in myelin and in the poor in myelin. However, there was a significant elevation in the number of these cells in the white rami on the 15th POD, where also occurs the most intense inflammatory reaction, in the gray rami it was found the opposite scenario, a significant decrease of the Schwann cells. With a lesser inflammatory reaction on the 45th POD and 180th POD, there was a progressive increase in the number of Schwann cells in the gray rami, maintaining lower levels than the white rami. The smaller number of Schwann cells found in the gray rami allows some speculations: (i) The elevated levels of collagen fibers and the small levels of reticular fibers in gray rami are suggestive of less regeneration, that might justify the smaller number of Schwann cells in these fibers; (ii) Knowing that the fibers of the gray rami are postganglionic, depending on the level of the cut selected, there is a greater possibility of studying the pre section segment in the gray rami communicantes, where the histological alterations caused by the injury occur in a mild way; (iii) The phagocyte action of the macrophages might be smaller in the gray rami due to the absence of myelin in the gray fibers, and, consequently, to the activation of the Schwann cells in the beginning of the degenerative process [8, 15].
Our experimental model does not allow the determination that neither the selected segments of the rami communicantes are in the pre- or post section segments, nor the quantity of the collagen and reticular fibers deposited during the process of cicatrization or regeneration would be enough to allow any function of the regenerated rami communicantes. Our data suggest that the regeneration of rami communicantes after the ramicotomy procedure occurs in a greater frequency than clinical recurrence of symptoms in humans.
Assuming that sympathetic regeneration is the main factor related to recurrence of symptoms after ramicotomy in humans, it is possible to develop some strategies to avoid it, such as using physical barriers between the sympathetic trunk and the rami communicantes after ramicotomy. A fragment of bovine pericardium, polytetrafluorethylene (PTFE) or a metallic clip could block the reconnection of the stumps.
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Abstract
1. Introduction
