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PubMed1989

Quantitative fluorimetric effects of leeching on a replanted ear.

Leeching is used to relieve venous congestion in microscopically replanted tissues. A quantitative description of the effects of leech application on tissue perfusion has not been reported. We report a case of an ear replantation in which venous congestion was consistently improved with leeching. In four instances, quantitative fluorimetry, which indicated impaired circulation and prompted leech application, showed restoration of normal circulation patterns following leech application. The fluorometric studies suggested that arterial inflow improved and venous congestion was relieved.

Adult↗

PubMed2003

Single-cell analysis reveals cell-specific patterns of expression of a family of putative voltage-gated sodium channel genes in the leech.

To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel alpha subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel alpha subunits, including the "S4 region" involved in activation, the "pore loops" responsible for ion selectivity, and the "inactivation loop" between the third and fourth domains, though the latter lacks the highly conserved "IFM" motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based "real time" RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system.

Amino Acid Sequence↗

PubMed1984

Quantitative effects of a neurotoxin upon serotonin levels within tissue compartments of the medicinal leech.

The serotonin (5-hydroxytryptamine, 5-HT) content of tissue compartments in the medicinal leech, Hirudo medicinalis, was measured by means of high-pressure liquid chromatography coupled with electrochemical detection (HPLC-EC). Each segmental ganglion contains 21.3 +/- 2.9 (9) pmol 5-HT [X +/- SEM (N)]. The pharynx contains 7.1 +/- 1.1 (9) pmol 5-HT/mg wet weight; the salivary glands 3.2 +/- 0.9 (10), ventral body wall 2.0 +/- 0.2 (11), and vasofibrous tissue 1.2 +/- 0.2 (11). The blood of hungry leeches contains 8.7 +/- 1.9 (7) nM 5-HT while that of well-fed leeches is 2.2 +/- 0.4 (6) nM. Leeches were injected with the cytotoxic analog of serotonin, 5,7-dihydroxytryptamine (5,7-DHT) producing selective lesions of the peripherally projecting serotonin-containing neurons, and which in turn abolished their feeding behavior. The serotonin content of the pharynx and ganglia of these toxin-treated leeches were lowered significantly. The serotonin levels within the body wall and salivary glands were not altered significantly by the toxin treatment, but the levels within the vasofibrous tissue and blood were elevated substantially.

5,7-Dihydroxytryptamine↗

PubMed1992

Differential effects of serotonin depletion on sensitization and dishabituation in the leech, Hirudo medicinalis.

The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.

5,7-Dihydroxytryptamine↗

PubMed1992

The role of matrix molecules in regeneration of leech CNS.

Extracellular matrix (ECM) molecules extracted from the leech central nervous system (CNS) provide substrates that induce extensive growth of processes of identified leech nerve cells in culture. Two ECM molecules, laminin and tenascin, have been identified. The laminin-like molecule has been purified and shown to be a cross-shaped molecule similar to vertebrate laminin with subunits of 340, 220, 180, and 160 kD. Purified laminin as a substrate induces rapid outgrowth of Retzius (R) and Anterior Pagoda (AP) cells in culture. The tenascin molecule has been partially purified. In electronmicrographs, leech tenascin, like vertebrate tenascin, has six arms of equal size joined in a central globule. Highly enriched fractions of leech tenascin induce rapid and extensive outgrowth of Retzius and AP cells in culture. Substrate molecules not only induce outgrowth of processes but also affect the growth patterns of individual nerve cells. Neurites are straight with few branches in laminin, but curved with profuse branches on tenascin. During regeneration of the CNS in the animal, laminin appears at new sites associated with growth cones. The appearance of laminin correlates with the accumulation of microglial cells. Thus, ECM molecules with growth-promoting activity for leech nerve cells in vitro appear to be involved in inducing regeneration and allowing the neurites to reconnect with former targets.

Animals↗

PubMed1993

Antennapedia-class homebox genes define diverse neuronal sets in the embryonic CNS of the leech.

Studies of the Antennapedia-class homeobox genes suggest that specific combinations of these transcription factors play a role in defining neuronal identities. We examined the expression of these genes in the leech Hirudo medicinalis, an organism well-suited for neurobiological research at the level of identified neurons. Leeches contain at least as many Antennapedia-class and related genes as insects do, despite the apparently lower complexity of the leech body plan. The CNS expression patterns of two Antennapedia-class leech homeobox genes (Lox genes) were examined in detail. Lox1 is expressed during early gangliogenesis in only one pair of transient neurons present in every segment (the Bipolar cells) and, at later stages of embryonic development, in 15-20 pairs of central neurons repeated in most segments. The monoclonal antibody Laz1-1 identified two pairs of Lox1-expressing neurons as the Bipolar cells and the L1 neurons. The Bipolar cells extended processes in the primordia of the longitudinal connective nerves and later degenerated. The L1 neurons were detected late in gangliogenesis and became stable neurons. Lox2 is expressed in an iterated set of neurons in the posterior two-thirds of the CNS. On the basis of cell body position and relative size, two pairs of Lox2-expressing cells were identified as the RPE-like neurons and the CV motor neurons. Other Lox genes are also expressed in segmentally repeated subpopulations of neurons. These neuronal subpopulations appear to be different from one another but partially overlapping. Different combinations of Lox genes that may be expressed in individual cells could in theory generate enough variability to specify all central neurons in a leech ganglion.

Animals↗

PubMed2006

Proteome modifications of the medicinal leech nervous system under bacterial challenge.

Once considered as lacking intrinsic immune mechanisms, the CNS of vertebrates is now known to be capable of mounting its own innate immune response. Interestingly, while invertebrates have been very useful in the interpretation of general vertebrate innate immunity mechanisms, only scarce data are available on the immune response of nervous tissue within this group. This study provides new data on the innate immune response of medicinal leech Hirudo medicinalis CNS. We identified several spots in 2-D gels of leech CNS proteins that showed specific changes following bacterial challenge, thus demonstrating the ability of the leech nervous system to mount a response to an immune stress. Protein identifications were based on comparison of sequence data with publicly available databases and a recently established leech ESTs database. The broad nature of the identified proteins suggests a clear involvement of cytoskeletal rearrangements, endoplasmic reticulum stress, modulation of synaptic activity and calcium mobilization, all during the first 24 hours of this response. Moreover, several of these proteins are specifically expressed in glial cells, suggesting an important role for glial cells in the immune response of the leech nervous system, similar to what has been observed in vertebrates.

Amino Acid Sequence↗

PubMed1993

Developmental regulation of a glycolipid epitope on actively extending growth cones and central and peripheral projections in the medicinal leech.

The monoclonal antibody, A2B5, that recognizes vertebrate gangliosides also recognizes embryonic cells in the medicinal leech (Hirudo medicinalis) in a spatially and temporally regulated way. Furthermore, A2B5-positive glycolipids could be isolated from embryonic leeches. Early in development A2B5 labeled axon tracts within the central nervous system coincident with the initial growth of axons, while later in embryogenesis, A2B5 also labeled the forming peripheral nerves. This central and peripheral staining disappeared during the latter third of embryogenesis. A2B5 also labeled the growing tips of an identified myo-organizing cell. This cell, the C-cell, projects an array of parallel processes and exhibits a discrete transition in the rate of growth cone extension (J. Jellies, and W. B. Kristan, Jr. 1991. Dev. Biol. 148, 334-354.). A2B5 failed to recognize the relatively non motile growth cones of the C-cell during early embryogenesis. The C-cell growth cones began to exhibit A2B5 labeling as they became more rapidly extending and this labeling persisted throughout the later motile phase of C-cell growth. In addition to its widespread distribution on embryonic (but not mature) cellular processes, A2B5 also labeled the mitotic profiles of dividing cells in all tissues. Thus, the A2B5 epitope may be presented intracellularly, or both intra- and extracellularly. When glycolipids were extracted from embryonic leeches, partitioned by elution from a silicic acid column, and analyzed using high-performance thin-layer chromatography, at least one of the major glycolipid bands was resorcinol-positive, consistent with presentation of sialic acid residues. The fraction enriched for the resorcinol-positive band was recognized by A2B5 on dot blots, as was ganglioside GQ1b. While potential mechanisms remain to be examined, on the basis of the distinct distribution of the A2B5 epitope and our finding of A2B5-positive glycolipids in leech embryos, we suggest that complex polar glycolipids may play a role in the extension of cellular projections in the medicinal leech.

Animals↗

PubMed2001

Validating Livanow: molecular data agree that leeches, Branchiobdellidans, and Acanthobdella peledina form a monophyletic group of oligochaetes.

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.

Animals↗

PubMed1999

Purification of granulin-like polypeptide from the blood-sucking leech, Hirudo nipponia.

A cysteine-rich (approximately 20%), low molecular weight (MW 6 kDa) polypeptide has been isolated from the Korean blood-sucking leech, Hirudo nipponia. From its amino acid composition and N-terminal amino acid sequence analysis, the new protein is similar to granulin (or epithelin), and so it has been named leech granulin. The leech granulin behaved as a thrombin inhibitor in the purification steps of size-exclusion, ion-exchange, chromatofocusing, and reverse-phase high-performance liquid chromatography. The leech granulin is an acidic peptide (pI 3.75) containing high cysteine residues with a unique sequence similar to granulins or epithelins isolated from other organisms. For the first time, a granulin-like polypeptide having thrombin inhibitory activity has been isolated from a leech species.

Amino Acid Sequence↗

PubMed1996

Mapping motor neuron activity to overt behavior in the leech. I. Passive biomechanical properties of the body wall.

As an initial step in constructing a quantitative biomechanical model of the medicinal leech (Hirudo medicinalis), we determined the passive properties of its body wall over the physiological range of dimensions. The major results of this study were: 1. The ellipsoidal cross section of resting leeches is maintained by tonic muscle activation as well as forces inherent in the structure of the body wall (i.e., residual stress). 2. The forces required for longitudinal and circumferential stretch to maximum physiological dimensions were similar in magnitude. Cutting out pieces of body wall did not affect the passive longitudinal or circumferential properties of body wall away from the edges of the cut. 3. The strain (i.e., the percentage change in dimension of different body segments when subject to the same force was identical, despite differences in muscle cross-sections. 4. Serotonin, a known modulator of tension in leech muscles, affected passive forces at all physiological muscle lengths. This suggests that the longitudinal muscle is responsible for at least part of the passive tension of the body wall. 5. We propose a simple viscoelastic model of the body wall. This model captures the dynamics of the passive responses of the leech body wall to imposed step changes in length. Using steady-state passive tensions predicted by the viscoelastic model we estimate the forces required to maintain the leech at any given length over the physiological range.

Animals↗

PubMed1991

Development of Trypanosoma fallisi in the leech, Desserobdella picta, in toads (Bufo americanus), and in vitro. A light and electron microscopic study.

The development of Trypanosoma fallisi of Bufo americanus from Algonquin Park, Ontario was studied by light and electron microscopy in blood culture, in its leech vector Desserobdella (= Batracobdella) picta, and in its toad host. In culture, bloodstream trypomastigotes transformed within one day to elongate epimastigotes which divided into rosettes. These gave rise to amastigotes, spheromastigotes, stumpy and elongate epimastigotes, and slender metacyclic trypomastigotes over a 4- to 6-day period. Development in the leech crop was similar to that in culture, with fewer amastigotes and no spheromastigotes observed. The stages in the leech were similar in size to their culture counterparts, except for metacyclic trypomastigotes, which were larger in culture. Culture and leech stages possessed a well developed cytostome-cytopharyngeal complex and prominent reservosomes. The kinetoplast of crop stages was small with a rectangular profile, but became larger and basket-like in the proboscis forms. Migration of trypanosomes to the proboscis appeared to depend on the rate of digestion of the bloodmeal. Flagellates in the leech were also characterized by the presence of intracellular microorganisms. Development of culture forms to mature stages in the toad was completed within 8 days postinoculation, with the organisms transforming into the typical "C"-shape with a large square kinetoplast. Natural infection of B. americanus was detected at 3 days postfeeding by D. picta and the resulting bloodstream trypomastigotes developed more slowly than inoculated cultured stages.

Animals↗

PubMed1985

Varicosity-associated antigens define neuropile subfields in the leech central nervous system.

A panel of twelve monoclonal antibodies raised against homogenates of leech nerve cords and four polyclonal antisera raised against purified neurotransmitters were used to label varicosities immunocytologically in the central neuropile of leech segmental ganglia. These immunoreactive varicosities occur in distinct patterns, some of which have a simple geometry. Three antibodies label immunoreactive varicosities distributed in a single dorsoventrally-oriented plane, two label varicosities distributed in lateral hemi-neuropiles (leaving void a central cephalocaudal passageway), and five label varicosities distributed throughout the neuropile. Six antibodies tested label varicosities across leech species, and five of these varicosity populations are distributed in patterns conserved across leech species. Immunocytologically-defined neuropile subfields do not correspond with previously identified histological and ultrastructural features of leech segmental ganglia. Analysis of immunocytologically-defined subfields is extended to include identification of sets of neurons which appear to project into these subfields, and to include an intracellular characterization of one of these neuron sets.

Animals↗

PubMed2000

The leech and the physician: biology, etymology, and medical practice with Hirudinea medicinalis.

The history of the word "leech" and the practice of leeching reveal interconnected social histories. We give the linguistic and medical histories of the word, and explore its biology and clinical history. Our historical account extends from the earliest known record of leeching to current research. Despite historical variation in its reputation as a therapeutic technique, leeching remains useful today in a number of applications. Further investigation may well disclose even more uses for the leech, particularly for its enzymes with anesthetic, anticoagulant, and antimetastatic properties.

Animals↗

PubMed1988

Antibiotic sensitivities of Aeromonas hydrophila cultured from medicinal leeches.

Increased use of medicinal leeches (Hirudo medicinalis) for the treatment of venous congestion in flaps and replanted parts has coincided with reports of soft tissue infections following leech application. We cultured the gullets of 20 medicinal leeches to re-examine the antibiotic sensitivities of Aeromonas hydrophila, the leech enteric organism associated with reported infections. These isolates reflected reported resistance to penicillin and first generation cephalosporins as well as sensitivity to gentamicin, tetracycline and chloramphenicol. Additionally, the cultures were sensitive to cefamandol, cefoxitin and two third generation cephalosporins (cefoperazone and cefotaxime). These findings suggest that cefamandol, cefoxitin and some third generation cephalosporins may have a role as perioperative antibiotics in replantation and flap surgery. These antibiotics might provide prophylaxis against Aeromonas hydrophila infection when leech use is required.

Aeromonas↗

PubMed1985

Serotonergic modulation of the feeding behavior of the medicinal leech.

Hungry medicinal leeches, Hirudo medicinalis, bite warm surfaces and ingest blood meals averaging 890% of their weight. Satiation lasts 12-18 months during which leeches avoid warm surfaces and will not bite. The segmental nervous system of the leech is distinguished by a population of neurons which contain serotonin (5-Hydroxytryptamine, 5-HT) at high concentrations. Some of these identified 5-HT neurons directly activate the effectors responsible for three physiological components of feeding: salivary secretion, bite-like movements and pharyngeal peristalsis. A localized warming of the lip is sufficient to initiate ingestion and synaptically excites anterior 5-HT cells into high frequency impulses or bursts. Distension of the body wall terminates ingestion and also hyperpolarizes these 5-HT neurons. Serotonin treatment produces hyperphagic behavior by the leech, while a specific pharmacological lesion of its 5-HT cell produces the anorexic behavior of satiation. This anorexia is transiently reversed by 5-HT treatment. Serotonin plays an obligatory role in the initiation and expression of leech feeding behavior by its differential modulation of central neuronal networks and peripheral glands and muscles.

Animals↗

PubMed1991

Serotonin is released from isolated leech ganglia by potassium-induced depolarization.

1. The quantities of serotonin that are released from isolated leech ganglia in vitro were measured with the sensitive neurochemical techniques of HPLC-EC. 2. Segmental ganglia were exposed to elevated concentrations of potassium that depolarize leech serotonin-containing neurons by approximately 35 mV per decade. 3. Each segmental ganglion released on average 0.20 pmol of serotonin during 10 min of incubation in a solution containing 64 mM K+. 4. The rate of serotonin release increased nearly four-fold to 0.74 pmol/10 min when ganglia were incubated in 120 mM K+. 5. The rates of ganglionic serotonin release in 120 mM K+ were quantitatively similar in these three, experimentally important species of leeches: Hirudo medicinalis, Macrobdella decora and Haementeria ghilianii. 6. Ionic substitution experiments with the divalent cations Mg2+ and Co2+ indicated that the release of serotonin from leech ganglia is mediated by a Ca2+ dependent process. 7. The serotonin-uptake blockers, imipramine and chlorimipramine, did not increase the amount of serotonin released in elevated potassium. 8. Vitally staining the identified serotonin-containing neurons with Neutral Red dye did not reduce the quantity of serotonin that was released from the ganglia in elevated potassium. 9. This study demonstrates the capacity of leech ganglia to release the neurochemical serotonin, and the rates of transmitter release increase with the degree of depolarization of serotonin-containing neurons.

Animals↗

PubMed2004

The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica.

The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determined using nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acid residues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kinds of globins by Clustal X revealed 22 invariant amino acids. The four globins were 26-33% identical. A striking feature of amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine is conserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus and Tylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T. heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), and human alpha and beta globins strongly indicated that the leech globins first separated from globin lineage of annelids.

Amino Acid Sequence↗