Degeneration and regeneration of neuromuscular junction architecture in rat skeletal muscle fibers damaged by bupivacaine hydrochloride

We evaluated the degeneration and regeneration of neuromuscular junctions (NMJs) on the extensor digitorum longus muscle of Fischer 344 rats between 4 h and 3 weeks after bupivacaine hydrochloride (BPVC) injection, which induces muscle fiber necrosis, using histochemical staining by acetylcholine esterase (AchE)-silver and electron microscopy. Degeneration of muscle fibers and NMJs was observed 4 h after BPVC injection. One week after BPVC injection, some terminal axons were almost completely retracted, and the level of basal lamina-associated AchE in some NMJ regions had gradually disappeared. At that time, the depression contained a few, mostly pit-like or elongated oval invaginations: the incipient junctional folds and some NMJs did not have any secondary junctional fold. By 2 weeks after the BPVC injection, secondary junctional folds began to develop: however, the number of secondary junctional folds was clearly less than that in normal NMJs. At 3 weeks when regeneration of muscle fibers was well advanced, the staining for AchE at the end-plates became stronger and better-defined. The volume density of mitochondria in the terminal area of the terminal significantly decreased upon BPVC-induced destruction of the NMJ, and the density reached the lowest value 24 h after BPVC injection. Significant changes in the ultrastructural features of the architecture of NMJs occurred in skeletal muscle fibers damaged by BPVC during both the degeneration and regeneration processes. The changes in the ultrastructural and morphological features of the NMJ architecture during the regeneration of degenerated muscle fibers resembled those that occur during the differentiation of normal muscle fibers.     

Rapid neuromuscular remodeling following limb immobilization

The effect of immobilization on endplate morphology of the rat soleus muscles was studied qualitatively and quantitatively. The endplate was visualized by light microscopic zinc iodide osmium (ZIO) staining and by electron microscopy. The soleus muscle was immobilized by pinning of ankle and knee joints at right angles for 5 days. Immobilized muscles were then compared to the contralateral side and to normal litter mates. After 5 days of partial disuse, muscle fibers atrophied and nerve terminal area increased in ZIO-determined measurements. Neuromuscular junctions (NMJs) of disuse muscle fibers visualized by electron microscopy exhibited greater amounts of degeneration than either contralateral or control NMJs. Degeneration consisted of nerve terminal disruption, exposed junctional folds, and postsynaptic areas which contained little or no postjunctional folds. Regeneration also occurred in the same NMJs, consisting of small terminals associated with large expansion of junctional folds, several small terminals occurring within the same primary synaptic cleft, and several axons wrapped by the same Schwann cell. These observations demonstrate, for the first time, that partial disuse for only 5 days produces muscle atrophy as well as denervation-like changes at the NMJ, which leads to terminal sprouting within the endplate area and remodeling.     

The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy

Summary Skeletal neuromuscular junctions (NMJs) of vertebrates were examined by scanning electron microscopy after removal of connective tissue components by HCl hydrolysis. In addition to the surface texture of NMJs, the subsynaptic organization of the sarcolemma was visualized in specimens in which nerve endings were detached from the muscle surface.A remarkable morphological variability between animal species was observed. The NMJs in the frog sartorius muscle consisted of longitudinal ribbon-like endings which fitted into a shallow synaptic gutter containing highly ordered cross-bands of junctional folds. The NMJs of the posterior latissimus dorsi muscle of the zebra finch were characterized by varicose swellings of the nerve endings which fitted into a round pit of the sarcolemma. NMJs in the sternothyroid muscle of the Chinese hamster consisted of thin ramified endings which were confined to an oval area on the muscle surface. The labyrinthine synaptic groove contained well-developed junctional folds without preferential spatial arrangement.The procedure used for the present study illustrates in great detail the terminal arborization of the motor nerve ending and the surface features of the subsynaptic sarcolemma. It may also allow quantitative study of the synaptic morphology of NMJs.     

Abnormalities in neuromuscular junction structure and skeletal muscle function in mice lacking the P2X2 nucleotide receptor

ATP is co-released in significant quantities with acetylcholine from motor neurons at skeletal neuromuscular junctions (NMJ). However, the role of this neurotransmitter in muscle function remains unclear. The P2X2 ion channel receptor subunit is expressed during development of the skeletal NMJ, but not in adult muscle fibers, although it is re-expressed during muscle fiber regeneration. Using mice deficient for the P2X2 receptor subunit for ATP (P2X2(-/-)), we demonstrate a role for purinergic signaling in NMJ development. Whereas control NMJs were characterized by precise apposition of pre-synaptic motor nerve terminals and post-synaptic junctional folds rich in acetylcholine receptors (AChRs), NMJs in P2X2(-/-) mice were disorganized: misapposition of nerve terminals and post-synaptic AChR expression localization was common; the density of post-synaptic junctional folds was reduced; and there was increased end-plate fragmentation. These changes in NMJ structure were associated with muscle fiber atrophy. In addition there was an increase in the proportion of fast type muscle fibers. These findings demonstrate a role for P2X2 receptor-mediated signaling in NMJ formation and suggest that purinergic signaling may play an as yet largely unrecognized part in synapse formation.     

Infraterminal spreading and extrajunctional expression of nicotinic acetylcholine receptors in denervated rat skeletal muscle

 Through the use of biotinylated-bungarotoxin and monoclonal antibodies, the nicotinic acetylcholine receptor (nAChR) was localized in the subneural apparatus of mammalian motor end plates of the flexor digitorum brevis muscle of the adult rat at the light and electron microscopic levels. Under normal conditions, nAChR was located in the primary post-synaptic membrane of the neuromuscular junction, and the depths of the junctional folds constituting the secondary post-synaptic membrane did not contain any nAChR. Up to 75 days after repeated transection of the related motor nerve (sciatic), there was no major alteration in the light-microscopic localization of junctional nAChR in the subneural apparatus, except for a moderate shrinkage and increased immunocytochemical reactivity of the subneural apparatus. At the electron microscopic level, however, immunocytochemical reactivity gradually occupied the entire extent of the secondary post-synaptic membrane, including the depths of the junctional folds, which exhibited extensive branching. In non-innervated portions of the muscle fibers, nAChR receptor appeared in a linear localization on the surfaces of denervated muscle fibers. This linear reaction was not continuous with the nAChR reaction of the motor end plates. It is concluded that denervation supersensitivity might not be due to spreading of junctional nAChR from the end-plate area, but rather to expression of nAChR in non-innervated portions of the muscle fiber and to the infraterminal (subsynaptic) spreading of nAChR into the depths of junctional folds. Received: 29 May 1998 / Accepted: 8 December 1998     

Effects of short-term denervation and subsequent reinnervation on motor endplates and the soleus muscle in the rat

The rat sciatic nerve was locally frozen, and changes in the nerve, motor endplates, and the soleus muscle were examined for up to 6 weeks by light and electron microscopy. The wet weights of denervated soleus muscles compared with contralateral values progressively declined to a minimum at 2 weeks after injury (60.7 +/- 2.5%) and began to reverse following 3 weeks. The sciatic nerve thoroughly degenerated after freezing. However, numerous regenerated myelinated and thin nerve fibers were observed at 3 weeks. They were considerably enlarged but still smaller than normal counterparts at 6 weeks postoperatively. Nerve terminals containing synaptic vesicles of endplates disappeared at day 1 and mostly reappeared at 3 weeks (about 70% of the endplates). All endplates examined were reinnervated at 4, 5, and 6 weeks. On the other hand, postsynaptic folds of muscle fibers seemed to be only slightly influenced by denervation or reinnervation. Ultrastructural alterations of myofibrils, in particular the loss of register, immediately appeared after denervation, spread progressively, peaked at 2 weeks, ameliorated following reinnervation, and became significantly normalized at 6 weeks after freezing. The proportion of type II fibers in the soleus muscle similary showed an increase and a decrease with a short delay in response to denervation and reinnervation, respectively. This study clearly demonstrated that the nerve supply affects the ultrastructural integrity of skeletal muscles. In addition, changes in the endplates and the soleus muscle evaluated in this study after short-term denervation are largely reversible following reinnervation.     

The morphological variability of neuromuscular junctions in the rat extraocular muscles: a scanning electron microscopical study

Six morphologically distinct types of neuromuscular junctions were identified by scanning electron microscopy in the rat extraocular muscles: two diffuse and four focal types. The diffuse junctions, spreading out extensively over the muscle fiber surface, were characterized by two types of varicose swellings (or terminal varicosities) of nerve endings. One type consisted of several ramifying nerve endings and shallow postsynaptic depressions with poorly-developed junctional folds. The other type consisted of a single axon and formed many synaptic contacts along the long axis of a muscle fiber. Round synaptic depressions facing the varicosities contained several junctional folds. The focal junctions, confined to an oval area on the muscle fiber surface, were characterized by the complexity and variability of their subneural apparatuses. Four different types of apparatuses, i.e., focal junctions, were found: 1) an apparatus consisting of labyrinthine gutters with numerous slit-like junctional folds, 2) apparatuses consisting of a large number (more than 20) of cup-like depressions with either a small number of pit-like junctional folds or 3) numerous slit-like ones, and 4) an apparatus consisting of a small number (about 10) of cup-like depressions with a few junctional folds. The findings indicate that the rat extraocular muscles contain six different types of muscle fibers.     

Low-intensity electrical stimulation ameliorates disruption of transverse tubules and neuromuscular junctional architecture in denervated rat skeletal muscle fibers

We determine the effects of direct electrical stimulation (ES) on the histological profiles in atrophied skeletal muscle fibers after denervation caused by nerve freezing. Direct ES was performed on the tibialis anterior (TA) muscle after denervation in 7-week-old rats divided into groups as follows: control (CON), denervation (DN), or denervation with direct ES (subdivided into a 4 mA (ES4), an 8 mA (ES8), or a 16 mA stimulus (ES16). The stimulation frequency was set at 10 Hz, and the voltage was set at 40 V (30 min/day, 6 days/week, for 3 weeks). Ultrastructural profiles of the membrane systems involved in excitation–contraction coupling, and four kinds of mRNA expression profiles were evaluated. Morphological disruptions occurred in transverse (t)-tubule networks following denervation: an apparent disruption of the transverse networks, and an increase in the longitudinal t-tubules spanning the gap between the two transverse networks, with the appearance of pentads and heptads. These membrane disruptions seemed to be ameliorated by relatively low intensity ES (4 mA and 8 mA), and the area of longitudinally oriented t-tubules and the number of pentads and heptads decreased significantly (P < 0.01) in ES4 and ES8 compared to the DN. The highest intensity (16 mA) did not improve the disruption of membrane systems. There were no significant differences in the _α1sDHPR and RyR1 mRNA expression among CON, DN, and all ES groups. After 3 weeks of denervation all nerve terminals had disappeared from the neuromuscular junctions (NMJs) in the CON and ES16 groups. However, in the ES4 and ES8 groups, modified nerve terminals were seen in the NMJs. The relatively low-intensity ES ameliorates disruption of membrane system architecture in denervated skeletal muscle fibers, but that it is necessary to select the optimal stimulus intensities to preserve the structural integrity of denervated muscle fibers.     

Ultrastructural study of neuromuscular junctions in experimental autoimmune myasthenia gravis (EAMG) in rabbits

Experimental autoimmune myasthenia gravis (EAMG) was produced in rabbits by the injection of acetylcholine receptor (AChR) protein from electric organ of Narkacion tokyonis with complete Freund's adjuvant, and the ultrastructural alterations of the neuromuscular junctions were studied. A lesion comparable to human myasthenia gravis could be observed in these animals. In acute EAMG, which showed a rapidly progressive severe paralysis 19 to 24 days after the first inoculation, distinct degeneration of the postsynaptic membrane was observed. Sometimes an irregular gap was found between the nerve terminal and postsynaptic sarcoplasm. In chronic EAMG with mild and prolonged muscle weakness, which appeared 23 to 54 days after the first inoculation, poorly developed junctional folds with little degenerative change were seen. By a morphometric analysis, some of the changes were detected even in a subclinical EAMG. In the presynaptic region, there was no ultrastructural alteration except that an increase of nerve terminal area was observed in chronic and subclinical EAMG. The pathogenesis of these alterations was discussed.     

Growth of regenerating skeletal muscle fibers in cats

Extensor digitorum longus (EDL) muscles of cats were grafted heterotopically or orthotopically, and either with or without prior denervation. The autografted muscles were studied at times from 4 to 518 days after grafting. Muscle weight, fiber cross-sectional area, and ultrastructural, histochemical, and biochemical characteristics of regenerating muscle fibers were determined. Prior denervation reduced the mass of muscle at the time of grafting, but had no significant effect on the characteristics of the regenerating muscles. Orthotopic and heterotopic autografts achieved similar recovery of structure. Mean fiber area reached control values. Differentiation into fiber types occurred, but compared to control muscles, autografts had fewer Type I and Type IIA fibers. Electron microscopic analysis of regenerating muscle fibers revealed centrally located nuclei, but otherwise normal ultrastructure. The bimodal distribution of Z-band width was consistent with differentiation into fiber types. Mean data of some morphological variables did not stabilize.     

Age changes of neuromuscular junctions in the extensor digitorum longus muscle of spontaneous thymoma BUF/Mna rats

BUF/Mna rats spontaneously develop thymomas and cause muscle weakness of hind legs at an advanced age. This rat strain has been recognized as a suitable animal model for human myasthenia gravis or related myopathies. To characterize the structural changes of neuromuscular junctions (NMJs) in BUF/Mna rats, subneural apparatuses (SNAs) of extensor digitorum longus muscles of young-adult (4-month-old) and aged (22- to 25-month-old) male rats were examined using scanning and transmission electron microscopy. The SNAs of NMJs in young rats consist of complex labyrinthine gutters with numerous slit-like junctional folds. SNAs in aged BUF/Mna rats, however, are characterized by: (1) a group of cup-like depressions with very wide slit-like junctional folds in relatively large muscle fibers (the major type), (2) the presence of slit-like folds on the flat sarcoplasm outside the cup-like depressions or on the protruded sarcoplasm, and (3) winding gutters or a small number of round depressions with poorly developed synaptic folds in small and medium-sized muscle fibers (the minor type). Since similar structural changes have been reported in dystrophic mice or normally aged rats, it is suggested that both the slowly progressing muscle atrophy and age-dependent turnover of muscle fibers may occur in the aged BUF/Mna rats. Received: 19 October 1999 / Accepted: 16 March 2000     

ULTRASTRUCTURAL STUDY OF NEUROMUSCULAR JUNCTIONS IN EXPERIMENTAL AUTOIMMUNE MYASTHENIA GRAVIS (EAMG) IN RABBITS

Experimental autoimmune myasthenia gravis (EAMG) was produced in rabbits by the injection of acetylcholine receptor (AChR) protein from electric organ of Narkacion tokyonis with complete Freund's adjuvant, and the ultra-structural alterations of the neuromuscular junctions were studied. A lesion comparable to human myasthenia gravis could be observed in these animals. In acute EAMG, which showed a rapidly progressive severe paralysis 19 to 24 days after the first inoculation, distinct degeneration of the postsynaptic membrane was observed. Sometimes an irregular gap was found between the nerve terminal and postsynaptic sarcoplasm. In chronic EAMG with mild and prolonged muscle weakness, which appeared 23 to 54 days after the first inoculation, poorly developed junctional folds with little degenerative change were seen. By a morphometric analysis, some of the changes were detected even in a subclinical EAMG. In the presynaptic region, there was no ultrastructural alteration except that an increase of nerve terminal area was observed in chronic and subclinical EAMG. The pathogenesis of these alterations was discussed. ACTA PATHOL. JPN. 35 : 621–629, 1985.     

The effects of age on atrophy and recovery in denervated fiber types of the rat nasolabialis muscle.

This study investigates the effects of advancing age on responses of nasolabialis muscle fibers to denervation and reinnervation. The nasolabialis is innervated by the facial nerve and is responsible for the whisking movement of the animal's large vibrissae. In young adult (3-month) and middle-aged (15-month) rats the muscle on one side of the head was denervated by crushing the facial nerve. At specific days postcrush, animals were sacrificed and thick sections of muscle were incubated to demonstrate cytochrome oxidase activity, a mitochondrial enzyme, which differentiated between red, white, and intermediate fiber types. The rate and extent of atrophy and recovery were evaluated using light microscope morphometric methods for which transverse fiber areas were measured and compared to fibers on the contralateral control side. There was an age-related delay in the time of functional return since older animals resumed normal whisking behavior 6 days later than the younger animals. In both age groups, white and intermediate fibers atrophied to the greatest extent and red fibers showed least atrophy. Despite the different responses of the fiber types to denervation, there was no age difference in the maximum degree of fiber atrophy within each fiber type. Age differences did occur in the rate of the denervation response since the middle-aged fibers consistently showed a more rapid significant atrophy than the young adult fibers. During recovery, older fibers may be limited in their ability to attain the size of fibers on the control side. The results indicate that through middle age, the process of advancing age increases the susceptibility of the nasolabialis muscle to denervation but does not alter the maximum extent of atrophy. The ability to recovery to normal fiber size, at least 2 months after denervation, is also age-related.     

The effects of age on atrophy and recovery in denervated fiber types of the rat nasolabialis muscle

This study investigates the effects of advancing age on responses of nasolabialis muscle fibers to denervation and reinnervation. The nasolabialis is innervated by the facial nerve and is responsible for the whisking movement of the animal's large vibrissae. In young adult (3-month) and middle-aged (15-month) rats the muscle on one side of the head was denervated by crushing the facial nerve. At specific days postcrush, animals were sacrificed and thick sections of muscle were incubated to demonstrate cytochrome oxidase activity, a mitochondrial enzyme, which differentiated between red, white, and intermediate fiber types. The rate and extent of atrophy and recovery were evaluated using light microscope morphometric methods for which transverse fiber areas were measured and compared to fibers on the contralateral control side. There was an age-related delay in the time of functional return since older animals resumed normal whisking behavior 6 days later than the younger animals. In both age groups, white and intermediate fibers atrophied to the greatest extent and red fibers showed least atrophy. Despite the different responses of the fiber types to denervation, there was no age difference in the maximum degree of fiber atrophy within each fiber type. Age differences did occur in the rate of the denervation response since the middle-aged fibers consistantly showed a more rapid significant atrophy than the young adult fibers. During recovery, older fibers may be limited in their ability to attain the size of fibers on the control side. The results indicate that through middle age, the process of advancing age increases the susceptibility of the nasolabialis muscle to denervation but does not alter the maximum extent of atrophy. The ability to recover to normal fiber size, at least 2 months after denervation, is also age-related.     

Ultrastructural studies of young and old mouse neuromuscular junctions

Summary The ultrastructure of the neuromuscular junction of young and old male CBF-1 mice was analysed both qualitatively and quantitatively. The age-related findings were similar in both the phasic extensor digitorum longus muscle and the tonic soleus muscle but more pronounced in the latter. Presynaptic terminals of old mice compared to young showed decreases in nerve terminal area, mitochondria and synaptic vesicles, but increases in smooth endoplasmic reticulum, coated vesicles, cisternae, microtubules and probably neurofilaments. On the postsynaptic side there were increases in complexity of junctional folds and subsarcolemmal vesicles, and the appearance of lipofuscin deposits. Occasional denervated postsynaptic regions were encountered in old neuromuscular junctions, but the predominant characteristics of aging changes were not those of denervation. Rather, a unique and uniform process involving most of the population of nerve terminals, possibly of physiologically adaptive significance, appears to occur with age in both phasic and tonic limb muscles.     

Scanning electron microscope study of neuromuscular junctions in different muscle fiber types in the zebra finch and rat

Examination by scanning electron microscopy revealed differences between neuromuscular junctions in the muscle fibers of the zebra finch (bird) and rat. The neuromuscular junctions between the anterior and posterior latissimus dorsi muscles of the zebra finch were compared. The junctions of the former, exclusively slow tonic fibers, were small and numerous along the long axis of a single muscle fiber. The synaptic depressions per junction were few. The junctions of the latter, exclusively fast twitch fibers, were large and consisted of more synaptic depressions than the former. Junctional folds were occasionally found in some depressions. The neuromuscular junctions between the extensor digitorum longus and soleus muscles of the rat were also compared. The former consisted almost entirely of fast twitch muscle fibers, whereas the latter consisted of both slow twitch fibers (75%) and fast twitch fibers (25%). The junctions in the extensor digitorum longus muscle were almost all labyrinthine gutters containing exclusively slit-like junctional folds. In the soleus muscle, two types of junctions were observed. One type was similar to that of the extensor digitorum longus muscle; the other was characterized by labyrinthine gutters containing sparse, narrow slit-like and pit-like junctional folds. We suggest from these structural differences of the subneural apparatuses that the junction of the fast twitch muscle is characterized by the subneural apparatus containing numerous slit-like junctional folds, and that of the slow twitch muscle fiber characterized by the apparatus containing sparse, narrow slit-like and pit-like junctional folds.     

Neuromuscular plasticity following limb immobilization

Summary The effects of immobilization on the ultrastructure of the rat neuromuscular junction of type I and type II muscle fibres were studied both qualitatively and quantitatively. Muscle fibre areas were measured as well The plantaris muscle was immobilized in a shortened position by applying a plaster cast for three weeks. Immobilized muscles were then compared to normal litter mates. Both type I and type II immobilized muscle fibres atrophied. Endplates from type II muscle fibres exhibited greater amounts of degeneration than type I endplates. Degeneration consisted of nerve terminal disruption, exposed junctional folds, postsynaptic areas which contained little or no postjunctional folds, and subjunctional sarcoplasmic masses. In addition to degeneration, the type II endplates also exhibited regeneration in the same endplate consisting of small terminals associated with large expanses of junctional folds, several small terminals occurring within the same primary synaptic cleft, and several axons wrapped by the same Schwann cell. These observations suggest terminal axonal regeneration. Our results demonstrate that limb immobilization produces muscle atrophy as well as denervation-like changes at the neuromuscular junctions which leads to terminal axonal sprouting and an ultrastructural remodelling.     

Postsynaptic membrane folds of the frog neuromuscular junction visualized by scanning electron microscopy.

We have used a scanning electron microscope to study the morphology of the postsynaptic folds in frog muscle. These folds were exposed by digesting muscles in collagenase or osmium tetroxide. During digestion the connective tissue and basement lamina were removed and the presynaptic nerve terminal pulled away from the muscle surface to reveal one or more concave junctional grooves, within which the folds in the postsynaptic membrane could be clearly seen. In places the folding patterns were more complex than could be inferred from single thin-section electron micrographs.The ease with which the postsynaptic folds of vertebrate muscles can be directly observed using this technique now makes it possible to investigate subtle changes in their structure during synaptogenesis, denervation or disease.     

Acetylcholine receptors at mature and aged mouse neuromuscular junctions

The density and distribution of junctional and perijunctional ACh receptors (AChR) were studied in young (8-12 months) and old (24-25 months) C57 mice to determine: (1) if increased amplitude of spontaneous postsynaptic potentials previously reported in old C57 muscle was due to increased junctional AChR; (2) if increased extrajunctional AChR would be found in association with previously reported nerve terminal complexity; and (3) if extrajunctional AChR was present as in disused or denervated muscle. Microdissection of individual muscle fibers combined with I125-alpha-bungarotoxin labeling, gamma counting, measurement of surface area, cholinesterase stains, and autoradiography were used to obtain the results. In both young and old mice there was a sharp gradient in AChR between the end-plate and the perijunctional region. End-plate AChR densities and total AChR per end-plate were the same at old and young end-plates, as were perijunctional values. Thus, neither end-plate nor extrajunctional AChR density changes with age. An increased mepp amplitude reported previously in old CB57 animals must be due to other factors. The perijunctional AChR in old mice show no changes characteristic of disuse or denervation, or those which might give rise to the observed nerve terminal complexity.     

Mitochondria-filled nerve endings around the terminal arteriole in the rat posterior cricoarytenoid muscle

We employed light and electron microscopy to examine the innervation of terminal arterioles in the rat posterior cricoarytenoid (PCA) muscle over a three-week period before and after unilateral denervation of the recurrent nerve. Observations showed an occasional single small-sized myelinated nerve fiber around the terminal arteriole in both normal and denervated PCA muscles. One axon after the last node of Ranvier of the small-sized myelinated nerve fiber, together with some nonmyelinated nerve fibers, was enwrapped by the Schwann cell and ran along the terminal arteriole, forming varicose swellings with numerous mitochondria along its course. In one instance, a small-sized myelinated nerve fiber in the denervated PCA muscle ramified into several branches containing numerous mitochondria. These nerve endings mainly existed in the adventitia, consisting of some layers of fibroblasts and collagen fibrils, and some of them were close to the vascular smooth muscle cells with only an intervening basal lamina. These findings suggest that the mitochondria-filled nerve endings may be sensory in nature, and function in response to the expansion and contraction of blood vessels caused by increased and/or decreased blood flow or intravascular pressure.