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PubMed1993

The persistence of hepatitis B antigen in the bloodmeal of the potential medicinal leech, Asiaticobdella buntonensis.

The persistence of the hepatitis B virus surface antigen (HBsAg) was used as an index of the survival time of this virus within the gastro-intestinal tract of the potential southern African medicinal leech, Asiaticobdella buntonensis. HBsAg was tested for in blood/faecal material at five intervals over 15 weeks. Samples from both the midgut and the rectum remained positive for the entire test period, although with decreasing strength. The results are compared with reports on other arthropods which indicate increasing antigen persistence with increasing body size. The findings implicate medicinal leeches as mechanical vectors of HBV and possibly of other medically important viruses, and argue against using leeches of suspect or unknown origin in the alleviation of venous congestion in failing microsurgical procedures.

Animals↗
PubMed1997

Clinical use of leeches in reconstructive surgery.

Although medicinal bloodletting with leeches is a practice that dates back 3500 years, leeches have been used in reconstructive surgery for only the past 35 years. In this article, the indications, mechanisms of action, and guidelines for using leeches in reconstructive surgery are reviewed.

Animals↗
PubMed1998

Basal acetylcholine release in leech ganglia depolarizes neurons through receptors with a nicotinic binding site.

The response of Retzius neurons, the main neuronal source of serotonin in the leech nervous system, to cholinergic agonists has been extensively investigated. In this study, we analyzed the effects of inhibiting the acetylcholinesterase (AChE) activity in the leech midbody ganglion on the electrophysiological activity of the Retzius neurons. Bath application of neostigmine and physostigmine (0.1-100 mumol l-1) produced, after a delay, a strong depolarization of the Retzius neurons with a dose-dependent amplitude and latency. The amplitude of this depolarization increased as the extracellular level of Ca2+ increased and decreased as the extracellular level of Ca2+ decreased. The response to neostigmine and physostigmine was inhibited by curare (100 mumol l-1), nicotine (10 mumol l-1), atropine (100 mumol l-1) and strychnine (100 mumol l-1), but was not affected by mecamylamine (100 mumol l-1) or hexamethonium (100 mumol l-1). Superfusion with solutions containing 100 mumol l-1 strychnine or atropine produced a progressive hyperpolarization of the Retzius neurons, while superfusion with 100 mumol l-1 curare did not. The hyperpolarization induced by atropine was inhibited in the presence of curare. Other neurons in the ganglion showed distinctive responses to the AChE inhibitors that were coincident with their responses to cholinergic agonists. The results suggest the existence of a basal level of acetylcholine (ACh) release in the leech ganglion that is powerfully counteracted by endogenous AChE activity. Under control conditions, this basal release appears to be sufficient to generate an ACh tonus that regulates the membrane potential of Retzius neurons. Since these neurons can support a sustained firing rate, which is dependent on the membrane potential, the results presented in this report suggest that the basal ACh tonus regulates the output of these neuromodulatory serotonergic neurons.

Acetylcholine↗
PubMed1998

Development of spontaneous and evoked behaviors in the medicinal leech.

ABSTRACT The ontogeny of behavior in an organism must reflect developmental events in the nervous system, and it thus provides a noninvasive measure of neuronal development. This approach may be particularly fruitful in the medicinal leech because the neuronal basis of several behaviors has been characterized in adult leeches, providing a rich background against which behavioral development can be interpreted. We have investigated the order in which behaviors arise during the period of embryonic development and have determined the time at which each behavior is first expressed. Some behaviors, such as lateral ridge formation, germinal plate bending, spiral twisting, and sidewinding, were produced spontaneously by embryos. Others, such as shortening, circumferential indentation, local bending, and elongation, occurred only when they were elicited by weak mechanical stimulation. Such stimulation rarely evoked a behavioral response in young embryos (at 45% of the time required for complete embryonic development, 45% ED), but by 80% ED embryos responded to nearly 100% of the stimuli presented. In embryos older than 50% ED, the behavior most frequently evoked by stimulation of the anterior end, the posterior end, or the rear sucker was shortening. Stimulation of the midbody usually evoked behavior other than shortening, illustrating that the body was behaviorally compartmentalized, at least in part. Some behaviors observed during embryogenesis are never seen in adult leeches. For example, in response to stimulation of the midbody, young embryos produced a behavior that we have called "circumferential indentation," whereas older embryos produced local bending, a response previously described for adults. The switch from circumferential indentation to local bending may signal the formation of new synaptic connections.

Animals↗
PubMed2005

By what mechanism do leeches help to salvage ischaemic tissues? A review.

The therapeutic use of leeches in medicine dates back to 50 b.c. and was cited by ancient authors. The medicinal leech, Hirudo medicinalis, has been used with increasing frequency during the past few years by reconstructive surgeons to help salvage ischaemic tissues. We aim to summarise the anatomy, physiology, and pharmacological mechanisms of action of leeches to provide reconstructive surgeons with a theoretical basis for their use.

Animals↗
PubMed2004

Celebrity with a neglected taxonomy: molecular systematics of the medicinal leech (genus Hirudo).

The medicinal leech is the most famous representative of the Hirudinea. It is one of few invertebrates widely used in medicine and as a scientific model object. It has recently been given considerable conservation effort. Despite all attention there is confusion regarding the taxonomic status of different morphological forms, with many different species described in the past, but only two generally accepted at present. The results of the phylogenetic analysis of a nuclear (ITS2+5.8S rRNA) and two mitochondrial gene sequences (12S rRNA, COI) suggest that the genus Hirudo is monophyletic. It consists, apart form the type Hirudo medicinalis and the East Asian Hirudo nipponia, of three other, neglected species. All of them have already been described either as species or morphological variety, and can readily be identified by their coloration pattern. The type species is in weakly supported sister relation with Hirudo sp. n. (described as variety orientalis) from Transcaucasia and Iran. Sister to them stands Hirudo verbana from southeastern Europe and Turkey, which is nowadays predominantly bred in leech farms and used as 'medicinal leech.' The North African Hirudo troctina is the sister taxon to this group of Western Eurasian species, whereas the basal split is between H. nipponia and the Western Palaearctic clade.

Animals↗
PubMed2004

Protein profiling of the medicinal leech salivary gland secretion by proteomic analytical methods.

Protein diversity of the high molecular weight fraction (molecular mass > 500 daltons) of salivary grand secretion of the medicinal leech Hirudo medicinalis has been demonstrated using methods of proteomic analysis. One-dimensional (1D) electrophoresis revealed the presence of more than 60 bands corresponding to molecular masses ranging from 11 to 483 kD. 2D-electrophoresis revealed more than 100 specific protein spots differing in molecular masses and pI values. SELDI-mass spectrometry analysis using the ProteinChip. System based on chromatography surfaces of strong anion or weak cation exchanger detected 45 individual compounds of molecular masses ranged from 1.964 to 66.5 kD. Comparison of SELDI-MS data with protein databases revealed eight known proteins from the medicinal leech. Other masses detected by proteomic analytical methods may be related to both modifications of known proteins and unknown biologically active components of leech saliva secretion.

Animals↗
PubMed2005

Collagen reorganization in leech wound healing.

BACKGROUND INFORMATION: Leeches respond to surgical lesions with the same sequence of events as that described for wound healing in vertebrates, where collagen is important for the development of tensions in healing wounds, functioning as an extracellular scaffold for accurate regeneration of the structures disrupted by surgical or traumatic actions. RESULTS: In surgically lesioned leeches, newly synthesized collagen is arranged in hierarchical structures. Fibrils can be packed and shaped to form cords or tubular structures, thus acting as an extracellular scaffold that directs and organizes the outgrowth of new vessels and the migration of immune cells towards lesioned tissues. In these animals, the general architecture of collagen fibrils, generated during tissue regeneration, shows similarities to both the structural pattern of collagen bundles and assembly processes observed in several vertebrate systems (fish scales, amphibian skin and human cornea). CONCLUSIONS: The production of extracellular matrix during wound healing in leeches is a surprising example of conservation of an extremely close relationship between the structure and function of molecular structures. It could be hypothesized that collagen structures, characterized not only by a striking structural complexity, but also by multifunctional purposes, are anatomical systems highly conserved throughout evolution.

Animals↗
PubMed1958

Comparison of the effects of eserine and neostigmine on the leech muscle preparation.

The relative potencies of eserine and neostigmine were determined on three preparations of cholinesterase. Eserine was found to be twice as potent as neostigmine on the pseudocholinesterase of horse plasma, half as potent on the true cholinesterase of cat central nervous system, but twelve times as potent on the true cholinesterase prepared from the leech body wall. On the leech preparation, about ten times smaller concentrations of eserine than of neostigmine were required to potentiate the acetylcholine response, but after removal of the anticholinesterase from the bath fluid, the potentiation persisted much longer after neostigmine than after eserine. The greater sensitivity of the leech muscle to eserine is fully accounted for by the fact that its cholinesterase is more sensitive to eserine than to neostigmine. The longer lasting potentiation after neostigmine on the other hand suggests that this anticholinesterase becomes more firmly attached to the cholinesterase receptors in this muscle than does eserine.

Acetylcholine↗
PubMed1999

Transformation of leech microglial cell morphology and properties following co-culture with injured central nervous system tissue

When the leech central nervous system (CNS) is injured, microglial cells migrate to the site of the lesion. It is possible that the injured CNS releases diffusible substances that alter the properties of microglial cells; to investigate this, microglial cells were cultured in the presence of injured or uninjured CNS tissue. Grown on Concanavalin A (Con-A), 75 % of microglial cells are rounded in shape and are avoided by growing neurites. However, when chains of leech ganglia with damaged connectives were cultured on Con-A next to microglial cells, many of the microglial cells changed their morphology. The number of rounded cells present decreased to 48 %, 4 % became spindle-shaped and 48 % had an intermediate form. In addition, the presence of crushed ganglionic chains allowed more growth of neurites across microglial cells than occurred under control conditions, although round-shaped microglia were still avoided by growing neurites. Similar changes in microglial cells were produced in cells plated on Con-A in the presence of conditioned medium from crushed ganglionic chains. Hence, a diffusible substance from injured CNS tissue caused the morphology of the microglial cells plated on Con-A to become more like that of microglia plated on laminin, on which only 22 % of the cells are rounded while the remainder are spindle-shaped and are readily crossed by neurites. Changes in morphology were not observed when microglial cells were cultured with frozen and crushed ganglionic chains or with uncrushed chains. These experiments demonstrate that substances released from damaged leech CNS cause microglial cells plated on Con-A to change their morphology and the way in which they interact with growing neurites.

Journal Article↗
PubMed1998

Basal acetylcholine release in leech ganglia depolarizes neurons through receptors with a nicotinic binding site

The response of Retzius neurons, the main neuronal source of serotonin in the leech nervous system, to cholinergic agonists has been extensively investigated. In this study, we analyzed the effects of inhibiting the acetylcholinesterase (AChE) activity in the leech midbody ganglion on the electrophysiological activity of the Retzius neurons. Bath application of neostigmine and physostigmine (0.1-100 &mgr;mol l-1) produced, after a delay, a strong depolarization of the Retzius neurons with a dose-dependent amplitude and latency. The amplitude of this depolarization increased as the extracellular level of Ca2+ increased and decreased as the extracellular level of Ca2+ decreased. The response to neostigmine and physostigmine was inhibited by curare (100 &mgr;mol l-1), nicotine (10 &mgr;mol l-1), atropine (100 &mgr;mol l-1) and strychnine (100 &mgr;mol l-1), but was not affected by mecamylamine (100 &mgr;mol l-1) or hexamethonium (100 &mgr;mol l-1). Superfusion with solutions containing 100 &mgr;mol l-1 strychnine or atropine produced a progressive hyperpolarization of the Retzius neurons, while superfusion with 100 &mgr;mol l-1 curare did not. The hyperpolarization induced by atropine was inhibited in the presence of curare. Other neurons in the ganglion showed distinctive responses to the AChE inhibitors that were coincident with their responses to cholinergic agonists. The results suggest the existence of a basal level of acetylcholine (ACh) release in the leech ganglion that is powerfully counteracted by endogenous AChE activity. Under control conditions, this basal release appears to be sufficient to generate an ACh tonus that regulates the membrane potential of Retzius neurons. Since these neurons can support a sustained firing rate, which is dependent on the membrane potential, the results presented in this report suggest that the basal ACh tonus regulates the output of these neuromodulatory serotonergic neurons.

Journal Article↗
PubMed2004

Aeromonas meningitis complicating medicinal leech therapy.

Medicinal leeches have an important and expanding role in medicine, but infection can complicate their use. We describe a unique case of Aeromonas meningitis associated with the use of leech therapy to salvage a skin flap after central nervous system surgery.

Adult↗
PubMed1996

Initial formation and secondary condensation of nerve pathways in the medicinal leech.

Invertebrates have proved to be important experimental systems for examining questions related to growth cone navigation and nerve formation, in large part because of their simpler nervous systems. However, such apparent simplicity can be deceiving because the final stereotyped patterns may be the result of multiple developmental mechanisms and not necessarily the sole consequence of the pathway choices of individual growth cones. We have examined the normal sequence of events that are involved in the formation of the major peripheral nerves in leech embryos by employing (1) an antibody directed against acetylated tubulin to label neurons growing out from the central nervous system, (2) the Lan3-2 antibody to label a specific population of peripheral neurons growing into the central nervous system, and (3) intracellular dye filling of single cells. We found that the mature pattern of nerves was characterized by a pair of large nerve roots, each of which branched into two major tracts. The earliest axonal projections did not, however, establish this pattern definitively. Rather, each of the four nerves initially formed as discrete, roughly parallel tracts without bifurcation, with the final branching pattern of the nerve roots being generated by a secondary condensation. In addition, we found that some of the nerves were pioneered in different ways and by different groups of neurons. One of the nerves was established by central neurons growing peripherally, another by peripheral neurons growing centrally. These results suggest that the formation of common nerves and neuronal pathfinding in the leech involves multiple sets of growth cone guidance strategies and morphogenetic mechanisms that belie its apparent simplicity.

Afferent Pathways↗
PubMed1996

Localization of the myomodulin-like immunoreactivity in the leech CNS.

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides.

Animals↗
PubMed1999

Glial hyperpolarization upon nerve root stimulation in the leech Hirudo medicinalis.

Hyperpolarizing responses in neuropil glial cells evoked by nerve root stimulation were studied in the central nervous system of the leech Hirudo medicinalis using intracellular recording and extracellular stimulation techniques. From a mean resting potential of -60.5 +/- 1.0, the glial membrane was hyperpolarized by -8.6 +/- 0.8 mV, via stimulation of the dorsal posterior nerve root in an isolated ganglion. Nerve root stimulation evoked biphasic or depolarizing responses in glial cells with resting potentials around -70 mV (Rose CR, Deitmer JW. J. Neurophysiol. 73:125-131, 1995). The hyperpolarizing response was reduced by the ionotropic glutamate receptor antagonist CNQX (50 microM) to 58% of its initial amplitude. In 15 mM Ca2+/15 mM Mg(2+)-saline the hyperpolarization was reduced by 44%. The hyperpolarization that persisted in high-divalent cation saline was not affected by CNQX. Bath-applied glutamate (500 microM) and kainate (2 microM) elicited glial hyperpolarizations that were sensitive to CNQX and 10 mM Mg2+/1 mM Ca(2+)-saline. The 5-HT-antagonist methysergide did not affect the hyperpolarizations evoked by nerve root stimulation. The results show that in the leech glial membrane responses to neuronal activity include not only depolarizations, as shown previously, but also hyperpolarizations, which are mediated by direct and indirect neuron-glial communication pathways. In the indirect pathway, glutamate is a transmitter between neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2000

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system.

We investigated the effects of ATP (adenosine 5'-triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose-dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na(+)-dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5'-diphosphate) > AMP (adenosine 5'-monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2-methylthio-ATP mimicked the ATP-induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8-SPT (8-(p-sulphophenyl)-theophylline) also depressed ATP-induced depolarizations and some ATP-insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP-induced depolarization requires activation of Na(+) channels or nonselective cation channels, whereas the ATP-induced hyperpolarization indicates activation of K(+) channels. ATP also increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), that is independent of Ca(2+) influx but reflects intracellular Ca(2+) release possibly triggered by IP(3) formation. ADP and AMP also increased [Ca(2+)](i), but were less efficient than ATP; adenosine and 2-methylthio-ATP did not affect [Ca(2+)](i). In view of the mobilization of intracellular Ca(2+), ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca(2+) signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized.

Adenosine Triphosphate↗
PubMed2000

Protein expression patterns of identified neurons and of sprouting cells from the leech central nervous system.

It has previously been shown that cephalic, segmental, and caudal ganglia from the medicinal leech show differences in their protein composition. Here we studied whether the neuronal reorganization that occurs in cultured segmental ganglia from the medicinal leech is accompanied by detectable changes in the protein expression pattern. Using silver-stained two-dimensional gels we showed that after 5 and 12 days in culture changes in the protein patterns can be detected in isolated ganglia. The changes observed in the two-dimensional gels occurred concomitantly with a sprouting of serotoninergic neurites and a decreased transmitter content of dopaminergic neurites as shown by using the glyoxylic acid condensation reaction. In addition, we present evidence that Retzius cells, which can be identified by their characteristic morphology and action potential waveform, exhibit biochemically unique properties with respect to their protein expression pattern.

Animals↗
PubMed2003

Progressive recovery of learning during regeneration of a single synapse in the medicinal leech.

The leech escape reflex-shortening of the body-can change with nonassociative conditioning, including sensitization, habituation, and dishabituation. Capacity for sensitization, which is an enhancement of the reflex, is lost when a single S-interneuron is ablated, but the reflex response itself remains. In the present experiments, the S-interneuron's axon in the living leech was filled with 6-carboxyfluorescein (6-CF) dye and cut with an argon laser microbeam (lambda = 488 nm). In contrast to sham-operated animals, axotomized preparations did not sensitize, reflecting the key role of the S-cell. By 2 weeks or more, S-cell axons had regenerated and reestablished synapses at their usual locations with neighboring S-cells. By 4 weeks, this restored the ability to sensitize to a level indistinguishable from that of controls, but an intermediate state of recovery was seen from 2-3 weeks after injury-a period not previously examined. The small capacity for sensitization among newly regenerated preparations was significantly lower than in sham controls but appeared higher than in animals whose cut S-cell axon had not regenerated its synapse. The results confirm the crucial role of the S-cell in sensitization. Moreover, full sensitization does not occur immediately upon synapse regeneration.

Animals↗