2.1 Marco Teorico
2.1.4 Mecanismos De Participacion Ciudadana
Forty Sprague-Dawley rats (200-250g) anaesthetized with Hypnorm (0.3ml/kg, i.m.) and Diazepam (2.5 mg/kg, i.p.) were given injections of Fast Blue (FB) (Dr. filing Gmbh, W est Germany) (5% w/v in ethylene glycol) into the skin, the gastrocnemius muscle, or the bladder, using a 30 g needle. For skin injections, the injections were made into three areas, the medial thigh (in the region innervated by the saphenous nerve), the
hairy skin of dorsal hindpaw (the region innervated by the superficial peroneal nerve) and the glabrous skin of ventral hindpaw (the region innervated by the tibial nerve). The dye was injected intradermally. Several injections were made within an area of up to 1 cm^. For muscle injections, the gastrocnemius muscle was exposed under sterile conditions, and multiple injections were made in the belly of the muscle taking great care to avoid injuring blood vessels. The wound was closed in two layers. For the bladder injections, the bladder was exposed in the abdominal cavity and multiple 1 pi injections were made into the bladder wall avoiding penetration into the bladder cavity or spillage into the peritoneum.
In all cases care was taken to avoid the dye tracking back out of the injection puncture site by giving the injection very slowly and by withdrawing the needle slowly. Six days after FB labelling, the animals were sacrificed and relevant ganglia dissected for cell culture.
A previous study in this laboratory (O'Brien et al., 1989) has shown that the optimal labelling in the dorsal root ganglia occurs 6 days after FB injection producing cells of high, and low intensity. Control experiments have demonstrated that the high intensity cells are target specific while the low intensity are due to systemic spread of the dye. (O'Brien 1989). The low intensity cells can be found in dorsal root ganglia outside the relevant dermatome, whereas high intensity cells are only present in relevant ganglia and such labelling disappears with lesions to nerves innervating the target site.
Relevant DRGs (L2-4, medial thigh; dorsal foot and hindpaw;
gastrocnemius; L ^-S j, L%_2, bladder), and thoracic ganglia (to check for systemic spread) from both sides of spinal cord were removed aseptically, and collected as seperate pools for each animal. The ganglia were then digested with 0.125% collagenase for 3 hrs and 0.25 % trypsin for 30 min, and mechanically dissociated through a flame-polished pasteur pipette. Cells were plated in 3.5 mm petri dishes at a density of 15,000- 20,000/dish and grown in F14 medium with 4% serum substitute Ultraser G (Gibco)
overnight. (Lindsay, 1988). The following day, capsaicin sensitive neurons in these cultures were identified by their ability to take up cobalt. No high intensity FB labelling was found in the neurons from thoracic ganglia .
The overnight-cultures were washed in calcium-free assay buffer (5 mM KCl, 2mM M gCl2, 12mM glucose, lOmM HEPES (pH 7.4), 137mM sucrose, 5.8mM NaCl ), then treated for 10 min in assay buffer containing 5mM C0CI2, with or without 10 pM capsaicin. The cultures were then washed quickly with assay buffer, developed in 1.2% ammonium polysulphide solution to precipitate the cobalt inside the cells, and fixed in 4% paraformaldehyde. (Hogan, 1982; Winter, 1987).
Cultures were examinated on a Nikon Fluophot microscope with filters appropriate for FB (excitation wavelengh 390-420 nm), and with transmitted light for cobalt uptake. Counts were made of high intensity FB cells (as specific labelling) and brown neurons as cobalt positive (Fig. 1), the staining of cobalt negative cells was light yellow which was just enough to be distinguished from background (Fig. 1).
Results
Examination of fixed cultures by FB filter under U.V. light showed that the intensity of FB labelling was similar to that observed in vivo. Some neurons were labelled seperately, in others the dye had leaked to satellite cells, producing an image like the sun surrounded by small planets. The specificity of FB labelling was maintained however, since even if a labelled cell was closely apposed to unlabelled cells in a cluster, the dye in the labelled cell did not pass to the unlabelled cells. The labelled neurons could be graded into two categories according to dye intensity-high and low. This was not related to cell diameter. The low intensity labelling was not usually found in cultures from skin-injected animals, but was seen in cultures from muscle or bladder-labelled animals. It is likely that the low intensity labelling is the result of systemic spread of FB
(O'Brien et al., 1989), as a few low intensity cells were found in the thoracic ganglia of muscle or bladder injected animals. Counts of these cells were omitted from the analysis.
The numbers of high intensity FB labelled neurons varied between individual animals. The proportion of these cells in cultures from skin- injected animals were on average 0.59%, those from muscle 0.64%, and bladder 0.9% .
Since capsaicin opens a non-selective cation channel, this raises a question whether FB will leak out from the open channel during cobalt uptake staining process. That this is not the case is indicated by the following observation. Upon experimental culture, the percentage of cobalt positive cells in total DRG neurons was 60± 1% (including FB labelled cells), whereas that of control cultures (no capsaicin) was less than 1%. (These figures are consistent with the finding by Winter, (Winter, 1987; W inter et al., 1988), who showed that approximately 50% of DRG neurons were capsaicin- sensitive in 2-5 day adult DRG cultures grown with nerve growth factor (NGF)). If the FB leaked out of the cells during the cobalt-uptake procedures, the number o f FB labelled cells in experimental dishes would be expected to be much less than in the control dishes. No significant difference in term of the number of labelled FB cells between experimental and control dishes was, however, found. This also shows that the cobalt sulphide does not obscure Fast Blue.
The data on the presence of cobalt uptake in high intensity FB labelled cells from skin, muscle and bladder are shown in Table 1. (individual data are shown in Table 1.1, 1.2, 1.3, 1.4, 1.5). In summary, there are regional differences between skin, skeletal muscle and urinary bladder, 20-30% of skin afferents, 40% of muscle afferents and 60% of bladder afferents were found to be capsaicin-sensitive. The proportion of capsaicin sensitive neurons in the retrogradely labelled population from the three target tissues were consistently different in each of the animals studied, with differences between three skin areas (hairy skin of dorsal hindpaw, glabrous skin of dorsal hindpaw and medial thigh) as well as between skin, muscle and bladder.
FIGURE 1
A 12 ,3 show the identical field. (A%) Phase- contrast micrograph shows DRG cells. (A2) Fluorescence micrograph shows a FB labelled cell. Note that cobalt staining decreases the background fluorescence but does not obscure FB labelling. (A3) Bright field micrograph shows that the labelled FB cell in (A2) is cobalt positive, that is capsaicin-sensitive. B % 2,3 show the same field. (B^) Phase- contrast micrograph of DRG cells. (B2) Fluorescence in the identical field shows a FB labelled cell. (B3) Bright field shows the FB labelled cell in (B2) is cobalt negative. Scale bar =50pm.1 ■
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