Fluoride-resistant Acid Phosphatase Activity Research Articles

Mapping of the fluoride resistant acid phosphatase (FRAP) activity in cervico-thoracic-lumbar spinal dorsal horn in rats

The fluoride resistant acid phosphatase (FRAP) is considered a biomarker enzyme of presence and function in nociceptive fibers. Its well described spinal dorsal horn (DH) superficial lamina location contrasts to its less known cervico-lumbar DH distribution. Thus, 15 male Sprague Dawley rats (300–400 g) were anaesthetized (thiopental 60 mg kg−1 i.p.) and laminectomized (C 1-L 5) to extract and fix (formaline 4% + NaF 1%) spinal cords. Cervical, thoracic and lumbar segments were coronally cut (1 mm slices) and micropunched (1 mmO) at the dorsal horn (both left and right). Samples were diluted in NaF 1% in saline (1000 μL: fragment) and homogenized. Protein concentration of the homogenate was determined by Bradford method to correct variation on tissue sampling and used to compensate enzymatic dilution. FRAP activity was determined by the Gomorri method and expressed as U gr prot−1 L−1. No differences were found between left and right DH FRAP activities (Mann-Whitney U Test, P > 0.05) in any metamera. DH segment values were: cervical 2.53 (2.67–2.40 U gr prot−1 L−1; mean ± 95% CI), thoracic 3.59 (3.96–3.21) and lumbar 2.42 (2.75–2.09). Thoracic FRAP activity was statistically higher (+45.3%) than those from cervical and lumbar DH segments (ANOVA; Ω2 = 0.29; F = 17.78; P = 0.000001; Levene test P = 0.000006). No significant correlation was found among cervicotoraco-lumbar FRAP activity. The increased FRAP activity for thoracic DH could be explained by the thoracic overlapped primary afferents and collateral fibers with cervical or lumbar input added to the basal thoracic FRAP expression.

Inflammation induced increase of fluoride resistant acid phosphatase (FRAP) activity in the spinal dorsal horn in rats

Fluoride-resistant acid phosphatase (FRAP) has been suggested as an enzymatic marker for nociceptive primary afferent terminals in the spinal dorsal horn, however there has not been demonstrated a direct functional relation between FRAP activity and an increased nociceptive transmission. For this purpose, we quantitated FRAP activity in the spinal dorsal horn of the rat in a heat-induced cutaneous inflammatory model. Male Sprague–Dawley rats anaesthetised with thiopental were separated in two groups where the left hindpaw was submerged during 60 s either in water at room temperature (control group) or in water at 60°C (inflammation group) which induce in this group a progressive hindpaw inflammation. After 8 h, the lumbar enlargement of the spinal cord was extracted, cut in slices and 1 mm micropunch fragments were obtained from the right and left dorsal horn. The activity of FRAP was determined using the Gomori colorimetric method and corrected by the protein concentrations. FRAP activity in the left dorsal horn was statistically higher than right dorsal horn in the inflammation group (3.05±0.54 versus 1.91±0.23 u/g per l; P<0.05). Also, FRAP activity from the left dorsal horn of the control and inflammation groups show a significant increase in the last group (3.05±0.54 versus 2.17±0.23 u/g per l; P<0.05). This results demonstrate that FRAP is not only an enzymatic marker for neuronal and fibre integrity of nociceptive primary afferents but also it is associated to the nociceptive afferent activation.

Fluoride-Resistant Acid Phosphatase (FRAP)-Positive Primary Afferent Central Terminals in the Mouse Substantia Gelatinosa.

The ultrastructure of the central terminals of nociceptive primary afferent (Cl terminals) neurons in the mouse substantia gelatinosa was studied by the cytochemical method using fluoride-resistant acid phosphatase (FRAP). Small roundish, slender, sinuous and large scalloped terminals showed FRAP reactivity. The FRAP reaction was seen as electron dense granular deposits between spherical synaptic vesicles and around the membrane of the synaptic vesicles. Especially, dark, small sinuous and large scalloped terminals showed intense FRAP reactivity. From the previous studies, these terminals were in accord with the capsaicin-sensitive Cl terminals. Furthermore, larger, roundish terminals with less packed spherical synaptic vesicles, many mitochondria and a few dense cored synaptic vesicles showed FRAP reactivity. Our recent results that Cll terminals are also sensitive to capsaicin support the FRAP activity of the Cll terminals in the present study. Thus, various types of FRAP-positive Cl terminals and some Cll terminals are considered to be central endings of the capsaicin-sensitive nociceptive primary afferents.

Developmental alterations in nociceptive threshold, immunoreactive calcitonin gene‐related peptide and substance P, and fluoride‐resistant acid phosphatase in neonatally capsaicin‐treated rats

This study examined the effect of neonatal administration of capsaicin on nociceptive threshold and the distribution of calcitonin gene-related peptide (CGRP), substance P (SP), and fluoride-resistant acid phosphatase (FRAP) in the dorsal horn of the spinal cord during the course of development (10 days to 12 weeks of age) in the rat. As early as 10 days of age, CGRP-like immunoreactivity was reduced in laminae I, II, and V, as well as in the bundles of fibers situated dorsal and ventral to the central canal. However, beginning on or about 6 weeks of age, the density of CGRP-like immunoreactivity in the superficial laminae and in the bundles dorsal and ventral to the central canal increased. Moreover, thick, nonvaricose CGRP-like immunoreactive fibers appeared in laminae III and IV. These recurring fibers were of primary afferent origin as demonstrated by their disappearance after multiple, unilateral rhizotomies. A similar age-dependent alteration in the density of FRAP activity was also observed. Although virtually absent at 10 days of age after neonatal administration of capsaicin, the density of FRAP activity increased in lamina II by 8 weeks of age. This activity disappeared after multiple, unilateral rhizotomies, indicating that the FRAP activity that reappeared was of primary afferent origin. Neonatal administration of capsaicin also reduced the density of SP-like immunoreactivity in the dorsal horn as early as 10 days of age, although the density of SP-like immunoreactivity showed some recovery after 6 weeks of age. However, unlike CGRP-like immunoreactivity or FRAP activity, the density of SP-like immunoreactivity in capsaicin-treated rats was not detectably altered by multiple, unilateral rhizotomies, indicating that it originated principally from intrinsic dorsal horn neurons. Age-dependent alterations in both thermal and mechanical, but not chemical, nociceptive thresholds were also observed in these same animals. Thus, tail flick latency, hot plate latency, and paw withdrawal threshold were maximally increased at 6 weeks of age, after which time thresholds declined to vehicle-treated values. In contrast, capsaicin-treated animals were uniformly insensitive to ophthalmic administration of capsaicin. The correspondence between developmental alterations in CGRP-like immunoreactivity or FRAP activity and in thermal and mechanical nociceptive thresholds is suggestive of a role of CGRP- or FRAP-containing primary afferents in thermal and mechanical nociception.

Distribution of small-diameter primary afferents containing oligosaccharide residues recognized by monoclonal antibody LA4 in the dorsal horn of the cat

Small-diameter primary afferents expressing oligosaccharide residues recognized by the monoclonal antibody LA4 constitute a newly described primary afferent population in the cat. LA4-immunoreactive primary afferents are found mainly in lamina II (LII) at all levels of the cat spinal cord. However, within LII immunoreactive fibers are most concentrated in outer LII and practically absent from the inner third of the lamina. In addition at sacral levels, a lateral bundle of immunoreactive fibers enters laminae V–VII. The distribution pattern and the type of oligosaccharide expressed by LA4-immunoreactive fibers suggest that they may be homologous to primary afferents shown in the rat to contain fluoride-resistant acid phosphatase activity. However, in the rat spinal cord the whole of inner LII is the main projection area of LA4-immunoreactive fibers (Alvarez et al., J. Neurocytol., 18 (1989) 611–629). This difference in the location of primary afferents within LII may reflect a species difference in the physiology of LII inner.

Cobalt-complex ATP enhanced regeneration in the dorsal horn of the rat spinal cord.

ATP, the energy source for axoplasmic transport, is indispensable for the transport of nerve growth factor (NGF). NGF regulates the regeneration of central processes of the primary sensory neurons, by means of transganglionic regulatory mechanisms. This central regeneration was investigated with the help of the histochemical detection of fluoride-resistant acid phosphatase (FRAP), one of the marker enzymes of the primary nociceptive neurons. Transganglionic degenerative atrophy (TDA) of the central terminals of primary sensory neurons was induced with sciatic nerve crush. Dynamics of central regeneration was studied in rats treated with a cobalt-ATP complex, and with commercially available Na-ATP, respectively, by means of histochemical detection of the restitution of FRAP activity in the Rolando substance. Disappearance of FRAP activity was complete on the 6th postoperative day in the medial two-thirds of the upper dorsal horn in segments L2-L6. The regeneration (i.e. replenishment of FRAP activity) began on the 14th day and was complete by the 31st day in animals treated with cobalt-ATP, while in the animals treated with Na-ATP the replenishment of FRAP activity began on the 20th day and was complete only by the 60th day. It is concluded that the cobalt-ATP-complex significantly enhances central regeneration.

Substance P-, somatostatin- and calcitonin gene-related peptide-like immunoreactivity and fluoride resistant acid phosphatase activity in relation to retrogradely labeled cutaneous, muscular and visceral primary sensory neurons in the rat : C. Molander, J. Ygge and C.-J. Dalsgaard, Neurosci. Lett., 74 (1987) 37–42

Substance P-, somatostatin- and calcitonin gene-related peptide-like immunoreactivity and fluoride resistant acid phosphatase activity in relation to retrogradely labeled cutaneous, muscular and visceral primary sensory neurons in the rat : C. Molander, J. Ygge and C.-J. Dalsgaard, Neurosci. Lett., 74 (1987) 37–42

Markers shared between dorsal root and enteric ganglia.

Although the bowel is known to contain intrinsic primary afferent neurons with mucosal projections these cells have not been identified. The current study was undertaken to determine whether carbohydrate differentiation antigens or enzymatic markers common to primary sensory neurons could be found in enteric neurons. Subpopulations of sensory neurons of rat dorsal root ganglia (DRG) can be identified by the immunocytochemical detection of lactoseries and globoseries carbohydrate differentiation antigens with monoclonal antibodies (MAbs) and by the histochemical demonstration of carbonic anhydrase (CA)- or fluoride-resistant acid phosphatase (FRAP) activities; therefore, these markers, and their coincident expression with neuropeptides, were studied in neurons of the rat small intestine. Subsets of enteric neurons were demonstrated by a MAb (1B2/1B7) recognizing greater than 45%, and by a MAb (alpha-SSEA-1) recognizing less than 0.1%, of DRG neurons, as well as by CA, but not FRAP activity. 1B2/1B7+ neurons were found in both the submucosal (approximately 46% of neurons) and myenteric plexuses (approximately 2% of neurons). Submucosal 1B2/1B7+ neurons with mucosal projections also displayed vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) but not substance P (SP) or calcitonin-gene-related peptide (CGRP) immunoreactivities. SSEA-1+ neurons were only found in the myenteric plexus, did not project to the mucosa (and thus are unlikely to be primary afferents), and were SOM+ or ENK+ but VIP- and NPY-. CA activity was intense in approximately 39% of the neurons of the submucosal plexus and in mucosal nerve fibers. Some (approximately 20%) of the submucosal CA neurons were also CGRP+, but VIP- and NPY-; therefore, MAb 1B2/1B7 and CA activity mark different nonoverlapping sets of submucosal neurons. Following the neonatal administration of capsaicin (50 mg/kg), 1B2/1B7 immunoreactivity was lost from all submucosal neurons; however, VIP immunoreactivity was not depleted from the cell bodies. Although it cannot yet be concluded that the MAb 1B2/1B7 or CA markers demonstrate the intrinsic sensory neurons of the gut, the presence in the bowel of both is consistent with the supposition that sensory neurons related to those of DRG are also found in the intestine. The functional and possible developmental significance of this relationship remains to be defined.

Neuropeptides in sensory perikarya projecting to the rat ovary

Afferent perikarya in dorsal root ganglia (DRG) at the T13 and L1 segmental levels projecting to the rat ovary were identified by utilizing the fluorescent retrograde tracer true blue (TB). Subsequently, TB-labeled ovarian afferent perikarya in DRG were examined for vasoactive intestinal polypeptide (VIP), substance P (SP), cholecystokinin-8 (CCK-8), neuropeptide Y (NPY), and somatostatin (SOM) immunoreactivity and for the presence of fluoride-resistant acid phosphatase (FRAP) enzyme activity. Of the ovarian afferent perikarya at the T13 and L1 segmental levels, 20.5% displayed VIP immunoreactivity, 23.8% displayed SP immunoreactivity, and 43.1% were immunoreactive for CCK-8. No ovarian afferent perikarya contained SOM or NYP immunoreactivity or FRAP activity. It is suggested that different neuropeptides may participate in modulation of specific ovarian functions.

Substance P-, somatostatin- and calcitonin gene-related peptide-like immunoreactivity and fluoride resistant acid phosphatase-activity in relation to retrogradely labeled cutaneous, muscular and visceral primary sensory neurons in the rat

The distribution of several peptides in cutaneous, muscular and visceral primary sensory neurons was investigated in the adult rat. The fluorescent dye Fast blue was applied to the proximal ends of transected saphenous (cutaneous), gastrocnemius (muscular) and greater splanchnic (visceral) nerves. Sections from corresponding dorsal root ganglia were incubated for simultaneous indirect immunocytochemical demonstration of calcitonin gene-related peptide (CGRP)-, substance P (SP)- or somatostatin (SOM)-like immunoreactivity (-LI) and Fast blue. In addition, the presence of fluoride resistant acid phosphatase (FRAP)-enzyme activity (-EA) in retrogradely Fast blue-labeled saphenous and gastrocnemius nerves was investigated by subsequent enzyme cytochemical analysis. The results revealed the presence of CGRP-LI, SP-LI, SOM-LI and FRAP-EA in cell bodies of primary sensory neurons which project to the saphenous and gastrocnemius nerves. CGRP-LI and SP-LI, but not SOM-LI, were found in splanchnic sensory neurons. The vast majority of the visceral sensory neurons were found to contain CGRP-LI.

Peripheral axotomy of the rat mandibular trigeminal nerve leads to an increase in VIP and decrease of other primary afferent neuropeptides in the spinal trigeminal nucleus

In the vasoactive intestinal polypeptide (VIP)-rich lumbosacral spinal cord, VIP increases at the expense of other neuropeptides after primary sensory nerve axotomy. This study was undertaken to ascertain whether similar changes occur in peripherally axotomised cranial sensory nerves. VIP immunoreactivity increased in the terminal region of the mandibular nerve in the trigeminal nucleus caudalis following unilateral section of the sensory root of the mandibular trigeminal nerve at the foramen orale. Other primary afferent neuropeptides (substance P, cholecystokinin and somatostatin) were depleted and fluoride-resistant acid phosphatase activity was abolished in the same circumscribed areas of the nucleus caudalis. The rise in VIP and depletion of other markers began 4 days postoperatively and was maximal by 10 days, these levels remaining unchanged up to 1 year postoperatively. VIP-immunoreactive cell bodies were absent from trigeminal ganglia from the unoperated side but small and medium cells stained intensely in the ganglia of the operated side after axotomy. These observations indicate that increase of VIP in sensory nerve terminals is a general phenomenon occurring in both cranial and spinal sensory terminal areas. The intense VIP immunoreactivity in axotomised trigeminal ganglia suggests that the increased levels of VIP in the nucleus caudalis are of peripheral origin, indicating a change in expression of neuropeptides within primary afferent neurons following peripheral axotomy.

A quantitative analysis of the interrelationships between subpopulations of rat sensory neurons containing arginine vasopressin or oxytocin and those containing substance P, fluoride-resistant acid phosphatase or neurofilament protein

In rat L5 dorsal root ganglia 50% of neurons contained arginine vasopressin-like immunoreactivity and 38% oxytocin-like immunoreactivity, the oxytocin entirely coexisting with the arginine vasopressin. Staining of alternate mirror-image sections with RT97 (an antibody to neurofilament protein, and a marker for large light neurons) and with arginine vasopressin antiserum showed that the two were entirely complementary, thus establishing arginine vasopressin as a marker for all small dark neurons. Mirror-image staining also showed that neurons containing substance P-like immunoreactivity and those containing fluoride-resistant acid phosphatase activity were each contained within the arginine vasopressin-positive population. Arginine vasopressin-like immunoreactivity was axonally transported in the dorsal root and (in greater quantity) in sciatic nerve. Arginine vasopressin-like immunoreactivity was present also in laminae I and II of the dorsal horn of the spinal cord and this reactivity was absent in animals which had been treated neonatally with capsaicin, suggesting that it was contained in primary afferent terminals. These results are discussed in terms of their implications for the classification of primary afferent neurons and of a possible physiological role for arginine vasopressin in these neurons.

Anatomical and cytochemical relationships of adenosine deaminase-containing primary afferent neurons in the rat

The distribution of adenosine deaminase-containing neurons and fibers in the spinal cord and medulla was examined and the relationship of dorsal root ganglia neurons containing this enzyme to those containing somatostatin, substance P, fluoride-resistant acid phosphatase (FRAP) and 5′-nucleotidase was determined using immunohistochemical and histochemical methods. In the spinal cord adenosine deaminase-immunoreactive fibers and neurons were confined to layer I and Ho. A similar localization of these was observed in the spinal trigeminal nucleus. In adult animals treated neonatally with capsaicin adenosine deaminase-positive fibers were totally depleted in layer IIo but only partially depleted in layer I. Anatysis of lumbar sensory ganglia revealed that small type-B neurons immunoreactive for adenosine deaminase were also immunoreactive for somatostatin but not substance P. In addition, adenosine deaminase-positive neurons lacked histochemical reaction-product for FRAP and exhibited the lowest activity of 5′-nucleotidase. Examination of the neuronal populations containing the two phosphatase enzymes showed that a proportion of neurons exhibiting 5′-nucleotidase activity were devoid of FRAP activity. It is concluded that dorsal root ganglia neurons immunoreactive for adenosine deaminase and somatostatin constitute a single subpopulation of type-B ganglion cells separate from those containing substance P or FRAP. It appears that the lack of coexistence of adenosine deaminase with either FRAP or 5′-nucleotidase cannot be attributed simply to a coexistence of the two latter enzymes since some 5′-nucleotidase-positive neurons lacking FRAP were also devoid of adenosine deaminaseimmunoreactivity. Insofar as these three enzymes may contribute to the regulation of transmission processes in primary sensory neurons, our results indicate a minimal functional relationship between adenine nucleoside and nucleotide degrading enzymes in these neurons. In addition, FRAP appears to have some functional independence from 5′-nucleotidase.

Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn

After peripheral nerve injury in rats, naturally occuring fluoride resistant acid phosphatase (FRAP) disappears from the substantia gelatinosa in that part of the dorsal horn in which the injured nerve afferents terminate. We have taken advantage of this fact to establish the spinal distribution of nerves innervating the skin of the hindlimb. The spinal map of the foot, the distal part of the lower lumbar dermatomes, is in the medial part of the substantia gelatinosa. More proximal skin of the thigh and the lower back maps laterally. The zone of disappearance of FRAP after sciatic nerve section did not shrink detectably within the first few months after injury, provided that regeneration of the nerve was prevented. After one year, however, central FRAP activity was at least partially restored. Secondary transection of the sciatic nerve eliminated the new enzyme and transection of neighboring nerves failed to do so. The restored FRAP activity therefore reflects renewed synthesis and transport of enzyme in still injured neurons, and not central sprouting of intact neighboring afferents.

Fine structure and fluoride resistant acid phosphatase activity of electron dense sinusoid terminals in the substantia gelatinosa Rolandi of the rat after dorsal root transection.

Fluoride resistant acid phosphatase (FRAP) activity of the rat substantia gelatinosa Rolandi is confined to electron dense sinusoid terminals under normal conditions. Transection of dorsal roots or removal of dorsal root ganglia results in a rapid degeneration of more than half of the electron dense sinusoid axon terminals. First signs of degeneration ensue 20 hours after surgery; at the 24 hours state osmiophilic degeneration bodies develop that are translocated into glial elements in the course of the second postoperative day. At the same time, light microscopically visible FRAP-activity of the Rolando substance disappears. Electron histochemical investigations reveal that decreased enzyme activity is due to degeneration of FRAP-positive terminals. It is concluded that FRAP-positive terminals, representing the majority of electron dense sinusoids in the Rolando substance, are dorsal root collaterals; the origin of non-degenerating FRAP-negative electron dense terminals remains unknown for the time being.