A fine structural demonstration that some benzopyrones act as vitamin P in the rat.

In rats fed a diet lacking flavonoids (but which had supplementary vitamin C) definite fine structural alterations were found in blood capillaries and tissues. These fine structural alterations were quite different from those reported in C-avitaminosis and imply a different deficiency. They were largely prevented by feeding the benzopyrones, coumarin or coumarin plus troxerutin, thus pointing to the specificity of the lesions. This implies that, for the rat, benzopyrones are vitamins and that vitamin C and "vitamin P"-deficiency states are qute distinct. In "P-avitaminosis" the basic lesion is the opening of some blood capillary endothelial intercellular junctions. Unlike in C-avitaminosis, the endothelial cells are intact, without pale, grossly swollen cytoplasms.

Benzo-pyrones in the treatment of chronic schizophrenic diseases.

Sixteen chronic schizophrenic subjects were treated for 3 months each with either a benzo-pyrone (Paroven/Venoruton, Zyma) or a placebo in a randomized, double-blind crossover trial. They continued to take their previous drug therapies. Therapeutic effects were measured by the Brief Psychiatric Rating Scale (BPRS), by self-assessment, and by assessment by a relative. Eleven patients completed the trial in relation to BPRS assessment. When on the active substance, as compared with the placebo, they showed a mean improvement of 27% (significant at the 1% level). Ten patients completed the trial in relation to the self and relative's assessments. There were improvements of 16% and 13%, respectively (significant at the 5% levels). Half the patients showed improvements on all the tests. Their improvements were 49%, 31%, and 21%, respectively. There was evidence that the active substance began to have an effect within 2 weeks. No side effects were observed with the active substance.

The quantitative morphology of interstitial tissue channels in some tissues of the rat and rabbit.

The water-rich phase (tissue channels) of the intersititial tissue in rat ileum, knee joint capsules, kidneys, and implanted Guyton's capsules was examined electron microscopically by the SEM of plastic injection models, and by TEM and HVEM of ferrocyanide and ferritin as tracers. It was shown that the channels do in fact exist, and are not just vacuoles. Quantitative estimations of their numbers and diameters were made. These agreed well with estimates made by other methods.

Variations in the numbers and dimensions of tissue channels after injury.

The dimensions and numbers of tissue channels were estimated, using transmission electron microscopy after filling them with ferri-ferrocyanide precipitate, in the subcutaneous tissue of mice. They were studied in the normal condition, and at varying times after a simple incised wound. After this injury they increased very greatly indeed in numbers and dimensions, being maximal at 1 week. Six months elapsed before they returned to normal. The hydraulic conductivity was calculated. It increased by 10(5) times at the maximum, when clinical oedema was present.

Benzo-pyrones in the treatment of lymphoedema.

Fifty clinical trials of 4 benzo-pyrones in the treatment of lymphoedema, by over 37 authors in 8 countries, are reviewed: 38 oral and 12 topical (11 and 6 of these, added to other therapies). Oral benzo-pyrones reduced oedema, symptoms (in almost all) and inflammation (SAI). These were significant and clinically important. There were no significant differences between arms and Grades 1 and 2 legs. Combining these 20 trials gave mean annual reductions of 55% of oedema (SE: 7.8%; 95% Confidence Interval: 40% to 71%) (p<0.001). Four trials of elephantitic legs gave 17% (4.8%; 7.6% to 27%), significantly less (p<0.01). Meta-analyses, tested by omitting non-double-blind or non-peer-reviewed trials, were robust. The greater the oedema, the greater the rate of reduction-lessening as time passed and the oedema reduced: annual reduction=37%x(79%) Period (p=0.01). Reductions varied with the molar dose (p=10(-8)): =0.10% (SE 0.013%) Dose (mg of coumarin or molar equivalent of other drugs). Topical coumarin also reduced oedema and symptoms. The results of some other therapies were improved by oral or topical benzo-pyrones 15% to 22% over a month and 0% to 78% over a year. These drugs are slow, but effective, cheap and convenient. Because of their slowness, compression garments are unnecessary. They were seldom used in trials. Side-effects are minimal. Only oral coumarin may cause idiosyncratic hepatitis (3 per 1,000). Topical coumarin does not, nor other benzo-pyrones.

A double-blind trial of calcium dobesilate in chronic venous insufficiency.

Calcium dobesilate was studied in a double-blind trial in 30 normal subjects and in 30 patients with chronic venous insufficiency (CVI). There were no significant alterations in the normal subjects. In patients with CVI the active drug gave significant improvements (p less than 0.01) in the measured volume of the foot and lower limb, in clinical tenderness (p less than 0.05), and in most of the symptoms (including feelings of heaviness, swelling, tiredness, and aching in the lower extremities). Even though the chronic venous insufficiency had existed for an average of fifteen years, a relatively short course of the drug caused considerable, and significant, improvements in a number of signs and symptoms of the disease.

A model of the factors affecting interstitial volume in oedema. Part II: Their effects at various abnormal steady-states.

A mathematical model has been used to investigate the alterations, in amount and importance, of the Factors controlling interstitial fluid volume in a number of steady-state oedemas. Steady-states were used because it is much easier to evaluate the effect of a specific Factor on oedema if all the others are constant. They are best compared if expressed in the same units: ml/min/100 g of tissue. The effects of lymphatic drainage on trauma are very important, as is the effect of tissue proteolysis on lymphoedema. Tissue proteolysis has minimal effects on the normal state and trauma; yet modelling its increase with benzo-pyrones markedly reduces oedema in both trauma and lymphoedema. Excess fibrosis greatly reduces lymphoedema. The effects of the individual Factors vary considerably between the different steady-states.

A model of the factors affecting interstitial volume in oedema. Part III: Partial derivatives and integrals at various volumes.

The effects of the individual Factors controlling interstitial volume vary between the different steady-states. Partial derivatives of volume with respect to each Factor show only the effects near the steady-state. In passing from one steady-state to another, these partial derivatives vary greatly. The total effects of a Factor are those of the integral of the partial derivative from the initial steady-state to the final one. The simplest way to measure a Factor's total effect is by setting it to zero and observing the difference in tissue volume when the model is perturbed. Thus, to find the effect of proteolysis in lymphoedema, for example, let proteolysis = 0 and compare the final volume in lymphoedema with that when proteolysis is allowed to occur. While the effect of proteolysis is important in lymphoedema, it is of minimal importance in trauma or normal condition. It is again important if proteolysis is increased by benzo-pyrones. There is little correlation between a partial derivative at a steady-state and the integral of this from normal to the state. In low-to-moderate oedemas, many Factors influence the fluid volume. When this volume becomes very large, all Factors except tissue hydrostatic pressure decrease in importance. Tissue hydrostatic pressure can increase indefinitely and is the ultimate reason that oedemas do not increase without limit.

A model of the factors affecting interstitial volume in oedema. Part I: Hierarchies, some new factors and their equations.

A mathematical model has been constructed to investigate the alterations, in amount and in importance, of the Factors controlling interstitial fluid volume in a number of steady-state oedemas. There are many so-called "Safety Factors" (at least 15), including a whole group concerned with regulating the amount of protein in the tissues (and thus tissue colloidal osmotic pressure). They are often in long hierarchies (of even up to 11 Factors), each influencing the next. Sometimes a Factor is recursive, i.e., it influences itself (either positively or negatively) via a Hierarchy of other Factors. The effects of some Factors differ in different Hierarchies, i.e., an increase in the Factor will increase VI via some of the Hierarchies and decrease it via some other Hierarchies. Which effect is most important varies with the Hierarchies and conditions existing at the time. The Factors are best compared if expressed in the same units: ml/min/100g of tissue. This is possible via appropriate multiplication factors derived from the relevant equations.

Are the initial lymphatics normally pulled open by the anchoring filaments?

The conformation of the walls of initial lymphatics was studied in oedema and during the filling- and emptying-phases of the initial lymphatic cycle in the mouse diaphragm and in the puppy jejunum. It was found that the anchoring filaments, attached to the endothelium, caused typical distortions of the vessels during oedema, but that these did not occur in normal tissue. This implies that these filaments do not pull them open during the filling-phase of the cycle, but that some other force causes them to fill.

Measuring and representing peripheral oedema and its alterations.

Correlation was very good between 1,500 simultaneous measurements of peripheral lymphoedema (arms and legs) by water displacement and by calculating volumes from circumferences, but in the legs "circumferences" gave only half the absolute amount of oedema when compared with "water displacement." For 150 arms, however, each method provided almost identical values for oedema. Arms when oedematous are fairly uniformly swollen; legs, on the other hand, are typically more oedematous distally. Circumference measurements accordingly include portions of nearly normal (i.e., minimally or nonoedematous) leg; water displacement by contrast measures only the oedematous, distal region. When only the circumferences of the lower legs were taken into consideration, the amount of oedema as measured by water displacement were almost identical. Nonetheless, measuring the proximal, more normal, or nonoedematous regions of the leg is critical for assessing treatment by physical methods (e.g., complex physical therapy). The various equations representing oedema can be greatly affected by errors in the initial, final or normal measured volumes. Relative errors differ as these variables alter. Many of the equations are non-linear, i.e., small alterations in one variable may produce widely differing results depending on the other variables. Problems in the use of an abnormal contralateral or "normal" limb as a reference point are discussed. The best equation to use in bilateral oedema is "Difference in Volume/Initial Volume"; in unilateral oedema the best equation is "Difference in Oedema/Normal". "Change in Oedema" i.e., "Difference in Oedema/Initial Oedema" is best derived from the Means of other equations.

A fine structural study of variations in protein concentration in lacteals during compression and relaxation.

The ratios of plasma proteins and ferritin (introduced into the gut) were determined between the jejunal lacteals and the interstitial channels in puppies. This was done by fine structural densitometry and the counting of molecules, respectively. In the normal state this ratio was approximately 2. In portions of tissue whose smooth muscle had been relaxed by atropine the ratio was approximately 1; in others, where the muscle had been contracted by Carbacol, it was approximately 3. These latter correspond approximately to the filling-phase and the emptying-phase of the initial lymphatic cycle, respectively. Thus the evidence was strengthened for an hypothesis, which holds that the filling is caused by the high effective colloidal osmotic pressure of the concentrated lymph.

The fine structure and functioning of tissue channels and lymphatics.

The fine structures of the tissue channels and the lymphatics are described. The two systems form part of the whole blood vascular-tissue channel-lymphatic complex. The fine functioning of this is very dependent on the structures of its individual components. In brief, the tissue-lymphatic system is a leaky swamp through which material flows due to the vagaries of adjacent pressure changes. It enters the lymphatics via holes in their walls: it is retained in them when the holes are closed.

Alterations of untreated lymphedema and it's grades over time.

Lymphedema is assumed to increase in amount and Grade with time. This work verifies that assumption, and may be helpful in persuading patients to undergo treatment and provides prognosis for likelihood of future disability. Before-treatment volumes of lymphedema/normal, Grades and durations' are compared in 231 postmastectomy arm, 74 primary leg, and 103 secondary leg lymphedemas. These were the first, consecutive, unilateral lymphedemas to receive treatment at 25 clinics whose therapists we had trained. Amounts of lymphedema increased with time, as did Grades. Arms increased more rapidly (p = 0.01) than secondary or primary legs, which did not differ from each other (linear regressions: 1.8 (0.34), 0.63 (0.20) and 0.68 (0.38) %/year, respectively). The Grades of primary lymphedemas increased more slowly than those of either secondary arms (p = 0.02) or secondary legs (p = 0.03), which did not differ from each other (regressions: 0.010 (0.0048), 0.038 (0.0063) and 0.032 (0.0048) Grades/year, respectively). Increase of lymphedema with Grade was less for secondary legs than for primary legs (p = 0.004) or arms (p = 0.009), which did not differ significantly (regressions: 9.9 (3.5), 35 (8.7) and 25 (3.4) %/Grade, respectively). Thus, arms increased more rapidly in size than primary or secondary legs; primary legs remained in each Grade longer--and got larger in them--than did secondary legs. This relative lack of fibrosis in primary lymphedema permitted greater amounts of edema and accorded well with histopathological studies. The increases described with duration were not caused simply by patient aging. Whereas both duration and the age at the onset of lymphedema were significant for the arms, only duration was significant for the legs. Even in the arms duration was much more important than age.

Electron microscopy of the effects of Unguentum lymphaticum on acute experimental lymphedema and various high-protein edemas.

Rats' legs and feet were studied by qualitative and quantitative electron microscopy, including mass densitometry of protein in the tissues and in the initial lymphatics. The tissues were either normal, or had been made edematous by lymphostasis, moderate burns, or dextran. It was found that Unguentum lymphaticum very greatly reduced the amount of edema in the legs with lymphostasis. Since the concentrations of plasma protein in the tissues and initial lymphatics, and its total amounts in the tissues were all greatly reduced, it appears that the cream's anti-lymphedematous activity is via a removal of the excess protein. Since the macrophages were greatly increased in number by the cream (and previous work shows that selectively poisoning these cells prevents much of the cream's effectiveness in lymphedema), very probably it is this increase in their numbers (and possibly their individual proteolytic activities) which is how the cream causes the removal of the excess protein--via an increased proteolysis. The cream also causes an increase in the amount of edema in the rat-foot (after all the injuries, including lymphedema); however the concentration and amount of protein in the tissues is reduced. This is consistent with an inflow of low protein fluid, caused by a vaso-dilatation of the blood microcirculation of this rather specialized tissue. All of these characteristics are very similar to those of many of the benzo-pyrone group of drugs.

The phylogeny of the fine structure of blood vessels and lymphatics: similarities and differences.

As animals increased in size, various modifications had to come into being to carry nutrients and waste around the body. Different phyla solved the various problems differently; sometimes there was convergent evolution. In invertebrates the endothelial cells are often widely separated from each other; permeability is limited by the pericyte layer; the reverse occurs in vertebrates. In primitive chordates small peripheral vessels often consist only of the basement membrane, and even this may be partly missing; the more centrally one looks, the more the endothelial cells become continuous. Fenestrae appeared first in the agnatha, but only become common in the elasmobranchs. Increased size and activity necessitated still larger blood hydrostatic pressure and increased blood colloidal osmotic pressures to balance this. Since the permeability of the vessels could not be reduced, much more protein (and fluid) had to leak to the tissues. So the lymphatic system had to evolve. This is first seen in the torpedoes and fully evolved in the bony fishes. However, the small venous vessels of the elasmobranchs have openable inter-endothelial junctions and other structures very similar to those of the initial lymphatics. Apart from the absence of fenestrae in lymphatics, or when this system is injected with a tracer, it is not always possible to tell them apart with the electron microscope. There are, however, various differences between them which will help to differentiate them.

A case of localised traumatic lymphoedema: observations concerning the obstruction of initial lymphatics and tissue channels by fibrin, and Menkin's hypothesis.

A case of acute lymphoedema is described. It was caused by a sharply localised, nearly circular, trauma. While there was minimal bruising, the trauma broke enough blood vessels to cause the blockage, by fibrin, of the initial lymphatics and tissue channels. This encircled a region, superficial to the deep fascia, and significantly prevented both the tissue fluid reaching the lymphatics and transport via the system. In addition, there was a short-lived, generalised lymphoedema, presumably caused by damage to the saphenous collecting lymphatics.

The effect of "Unguentum lymphaticum" on acute experimental lymphedema and other high-protein edemas.

A cream, Unguentum lymphaticum, which has been shown to be effective (clinically and experimentally) in lymphedema, was tested in dextran and burn edemas and acute lymphedema in rats. It was very effective indeed in lymphedema, completely preventing the 36% increase in the volumes of the legs found with in those treated with its drug-free base. This protection was much less if the macrophages were selectively poisoned with silica, and the edema reached maximum volume much more rapidly. This shows that most of the cream's activity against lymphedema is via an increase of the normal proteolysis by macrophages, and also confirms that these are of considerable importance in limiting lymphedema (and other high-protein edemas). Curiously, the cream slightly increased the edema of the feet in acute lymphoedema, and also in dextran and burn edema, although other workers did not find this with histamine and egg albumin. This, and other evidence, suggests that another part of its action is vaso-dilatory - at least to rat-feet. Obviously it has many actions. While they should be investigated, the most important thing is that this cream offers a relatively cheap therapy (perhaps in combination with others: perhaps alone) for the nearly 300,000,000 people who suffer from lymphedema.

Treatment of lymphedema by complex physical therapy, with and without oral and topical benzopyrones: what should therapists and patients expect.

Using previously presented data, from an open multi-centered trial, the Medians and Third Quartiles were calculated for the overall edema reductions the "average" patient received from an "average" therapist using Complex Physical Therapy (CPT) on 628 lymphedematous limbs. These data provide therapists and patients a general guideline as to the minimal reductions which can be expected in three quarters of patients from average therapists, using CPT with or without the administration of benzo-pyrones. Benzo-pyrones were considered separately: oral (given for three months before and for 11 months after the course of CPT) and topical (during and after CPT). Depending on which measure of edema and its alterations were used, these reductions increased those from CPT alone from 130% to 200% and, after one year, between 150% to 300%. Whereas the effects of oral and topical benzo-pyrones were not statistically different, their combined usage was more effective than use of either alone.

The effects of calcium dobesilate on acute lymphedema (with and without macrophages), and on burn edema.

Calcium dobesilate ("Doxium", O.M. Laboratories, Geneva) was tested in two high-protein edemas. While at a high dose level it had no effect on burn edema of the ratfoot, it did at a low dose. It greatly reduced (to 26%) acute lymphedema in the rat thigh (although not in the foot). Electron microscopy confirmed these findings, and showed that the drug increased the number of macrophages in the tissues and reduced the protein concentrations. If, however, the macrophages were destroyed (by silica) this effect on acute lymphedema was lost (as with benzo-pyrones). Calcium dobesilate also reduces high-protein edemas in other models, and in many ways its actions resemble those of benzo-pyrones. Hence it is suggested that this substituted benzene ring may be the basic structure responsible for this action in high-protein edema, and that the pyrone ring (and any side-chains) in the benzo-pyrones are not essential to their enhancement of proteolytic activity by macrophages.