Alimentarni trakt i pankreas

                   ARCH GASTROENTEROHEPATOL 2001; 20 ( No 3 – 4 ):

The role of SMS 201-995 as an experimental model in the intestinal ischemia-revascularisation syndrome

Uloga SMS 201-995 na sindrom intestinalne ishemije-reperfuzije na eksperimentalnom modelu

( accepted  November 20th, 2001 )

¹Fernando Fuertes- Guiró, ²Joan Viñas Salas, ³Corrado d’Urbano

¹International University of Catalonia, Barcelona, Spain,

² Lleida Medical School. University of Lleida. Spain,

³ Department of Emergency Surgery, Provincial General Hospital

of Milan, Milan,  Italy.

Address correspondence to: Professor Fernando Fuertes-Guiró, MD, PhD, PhD ( Eur )

                                                 International University of Catalonia

                                                 Calle Gomera s/n

                                                 E-08190

                                                  Sant Cugat del Vallés

                                                Barcelona, Spain

                                                FAX: +34 93 5042001

                                                E-mail: [email protected]
 

ABSTRACT

AIMS: The main purpose of this research is to verify whether the SMS 201-995 as the only therapeutical option, and using a research protocol, has beneficial effects on the ischemia-revascularization syndrome.

MATERIAL AND METHODS: Sixty four male Wistar rats (32 for the control group and 32 for the treated group) were randomized in four studies: 1. Biochemical study (36 hours of observation), obtaining preoperative and postoperative quantitative data (Na, K,  Cl, and bicarbonate intestinal serum content); 2. Histological study: a) microscope, using 4 stains and a score for the evaluation of the lesions; b) ultrastructurally with scanning electron microscope, observation of objective variations on the surface of villi; 3. Survival parenteral hydration (0.25 mL/100 gr. weight of Ringer lactate and 0.1% of albumine) with yugular cannula, testing the Pi, the BUN and the total survival on constant periods of time; 4. Survival  without hydration, observing only the total survival. The SMS 201-995 dose was 10 ug/100gr./8h, subcutaneously.

RESULTS: The treated animal group maintained serum levels of Na, K, Cl and bicarbonate closer to the basal levels than the control groups, being the Na, K, Cl and bicarbonate significantly different. The 4 ions concentration and content of the guts and the volume of intestinal content were significantly lower on the treatment group. The  histological lesions of the guts was also better among the treatment group. The treated group- with or without hydration- increased the survival level. The quantitative grade of variation and the velocity of variation of the BUN and the Pi in blood was significantly lower among the treatment group.

CONCLUSIONS: The SMS 201-995 or octreotide, as the only  pharmacological option during the revascularization period on a controlled ischemia-revascularization syndrome, reduces the intestinal liquid retention, mantains the absorption- secretion intestinal process ( which  has a positive effect in the intravascular space), and reduces the severity of the lesions  in the gut. The cytoprotective effect, the protection  on a catabolic stress situation and the capacity to maintain a reasonableb level of volemia should permit to prolonge the animal’s survival.

Key words: octreotide, ischemia-revascularization, gut, survival, intestinal absorption- secretion.

SAZETAK

CILJEVI: Glavni cilj ispitivanja je da se utvrdji da li SMS 201-995 kao jedino terapijsko sredstvo u istrazivackom postutku ima povoljan efekat na sindrom ishemije-revaskularzacije.

MATERIJAL I METODE: 64 Wistar miseva ( 32 kontrolna grupa, 32 lecena grupa ) su bili su metodom slucajnog izbora ukljuceni u 4 ispitivanja: 1.Biohemijska studija ( posmatranje tokom 36h ) koja je podrazumevala pre- i postoperativno pracenje Na,K,Cl,HCO₃ u serumu iz creva; 2.Histoloska studija: a)mikroskopski pregled koji je ukljucivao 4 vrste bojenja i stepenovanje crevnih ostecenja; b)ultrastukturno ispitivanje “scanning” elektronskim mikroskopom u cilju utvrdjivanje promena crevnih resica; 3.Tokom primene parenteralne hidracije preko jugularne kanule u cilju prezivljavanja ( 0.25 ml/100gr  tezine  Ringer lactata and 0.1%  albumina ) mereni su P i azotne materije ( urea ) i duzina prezivljavanja u definisanom vremenskom periodu; 4. Merenje prezivljavanja bez hidracije. Doza SMS 201-995 je bila 10 ug/100gr/8h potkozno.

REZULTATI: U lecenih zivotinja odrzavani serumski nivoi Na,K,Cl, HCO₃ su bili blizi bazalnim vrednostima nego u kontrolnoj grupi. Koncentracija ova 4 jona, njihov sadrzaju u samome crevu kao i zapremina crevnog sadrzaja  su bili znacajno nizi u lecenoj grupi zivotinja. Crevne histoloske promene su bile takodje  blaze izrazene u tretiranih zivotinja. Lecene zivotinje, hidrirane i nehidrirane su duze prezivljavale. Kvantitativno izrazeno stepen i brzina varijacija  promena ureje i P u krvi su bili manji u oktreotidom tretiranih miseva.

ZAKLJUCI: SMS 201-995 ili oktreotid, kao jedino primenjeno farmakolosko sredstvo tokom perioda revaskularizacije na eksperimenatalnom modelu sindroma ishemije-revaskularizacije, je smanjivao zadrzavenje tecnosti u crevu, odrzavao je procese crevne apropscije i sekrecije ( sa pozitivnim efektom na intravaskularni prostor ) i smanjivao je tezinu crevnih ostecenja. Citoprotektivno dejstvo, zastita od katabolickog stresa i sposobnost odrzavanja volemije su bili u osnovi produzenja prezivljavanja zivotinja.

Kljucne reci: oktreotid, ishemija-revaskularizacija, crevo, prezivljavanje, crevna apsorpcija-sekrecija.

INTRODUCTION.

            The polypeptide hormone somastotatin has different and multiple physiological functions in the human body. Initially found to be a powerful inhibitor of the  growth hormone in the hypothalamus, somastotatin also fills other important functions in different areas of the body.

Somastotatin inhibits gastrointestinal secretion  by reducing the secretion of Cl, Na and water into the intestinal lumen and increasing the absorption of water and electrolytes from bowels (1,2). Other physiological functions include the following: 1) intestinal blood flow by inhibiting or stimulating intestinal neurons, 2) it produces an analgesic effect due to its affinity with the opioid receptors,  3) it renews the intestinal mucosa through its effects in the intestinal bowel crypts, 4) and it releases or inhibits other gastrointestinal hormones such as vasoactive intestinal peptide (VIP) (3,4,5). However, the clinical benefits of this hormone are limited due to its short plasma half-life. Recently synthetic analogues with the same biological effects and longer half-lives have been developed one of these molecules is SMS 201-995.

The ischemia-revascularization intestinal syndrome is a time-depenent, well-defined pathological entity within a patophysologic frame based in two specific stages; the ischemic stage or hypoxemia and the revascularization stage or the reintroduction of oxygen into the ischemic tissues. As a result, the intestinal function of absortion-secretion, the blood flow, the cellular integrity, the mucosa wall, its motility and the hormonal function of the intestinal tract ara impaired.
Recently there has been a great number of research studies on the synthetic analogue, the octreotide, and its role in the ischemia-revascularization syndromes (6,7). On the another hand, other studies focused on the physiopathology of this lesion and on the role of the SMS 201.995 in this pathology including: cytoprotective effects, inhibitory effects on the pancreatic proteases, and effects on the intestinal acid-based equilibrium (8,9,10).

Our hypothesis is that the SMS 201-995 or octreotide, a synthetic analogue of the somatostatin has beneficial effects on acute ischemia-revascularization syndrome and its pathophysiologic consequences. An experimental study has been designed based on biochemical, clinical, histological and survival data, in a protocol in which the octreotide is the only pharmacological and therapeutical option.

MATERIAL AND METODS

Biochemical clinic study. (From 20 animals, 10 had been treated with octreotide and 10 with placebo). The study consisted of four stages: Stage 1. The animals were housed for a week; Stage 2. First surgical operation was performed: an ischemic lesion was performed by temporary ligation(90 min) with  a clamp of the craneal mesenteric artery at its bifurcation of the aorta . Before the ligation, basal blood rates of Na, Cl, K and bicarbonate were measured; Stage 3. The observation stage: 36 hours of observation during which the octreotide /Italfarmaco SpA, Milan, Italy), was administered subcutatenously, 10 ug/100 gr. /8h diluted up to 2 mL of saline, or placebo, 2 mL of saline only; Stage 4. A second surgical operation: the quantitative data (venous concentration of Na, Cl, K and bicarbonate) were recorded. After removal of the whole  length of the small bowel, the intestinal volume before and after centrifugation of the guts and the intestinal concentration of Na, Cl, K and bicarbonate were recorded. From this data the total intestinal content of the electrolytes were obtained. Blood samples were taken from portal vein. All samples were frozen after centrifugation to –80ºC and analized using the same technique and reactives.

Histological and ultrastructural studies. ( 24 animals). The intestinal content of 20 animals corresponding to the precedent study was emptied. A segment of ileum was fixed in formol 6 % dehydrated and immersed in paraffin, cuts of 5 um were obtained and stained with hematoxilin of Harris-eosin, hematoxilin of Harris-floxin, PAS Schiff A-B and trichromic with blue amiline according to the Masson tecnique and observed on an optic microscope.

The remaining 4 rats followed the same protocol and split in two groups of 2 (control and treated) but no quantitative nor qualitative data was obtained. Once the intestinal segment was removed and fixed in glutaraldehide 2.5% and phosphate tamponade 0.1M,a specific treatment was performed before being observed on the scanning microscope. This treatment involved cleaning with tampon phosphate 0.1 M, dehydrating with acetone at increased concentrations, drying with CO2 according to the critical point dryer method and coating with coal of 400 A thick and gold. The readings of the samples on the optic  microscope were quantified with a score based on variables with increased according the severity of the lesions:

            • Villi, according to the classification of Chiu and alies (11)

• The integrity of the musocal barrier.

• The type and quantity of inflammatory infiltrate and oedema in

  the lamina propia, submucosa, muscular, serosa layers.

• Dilatation of intrapariental vessels.

The readings of the samples on the scanning electronic microscope (Zeiss DMS 940 A) were performed on an objective- comparative evaluation based on four parameters: villi-number, size and shape-, apix of villi, stage and type of mucosal secretion and bacterial flora and structure of the brush border.

Survival studies with parenteral hydration. ( 20 rats, 10 with octreotide, 10 with placebo). Three stages were carried out: Stage 1. The animals were housed for one week; stage 2. A surgical operation similar to the lesion produced by the temporary ligation of the artery on the previous study, catheterization of the external jugular vein and measuring of the basal rates of inorganic phosphorus and plasmatic urea; stage 3. The period to expiration of the animal (cardio-respiratory arrest).During this last stage, the venous concentration of inorganic phosphorus and the urea in blood samples of the tail veins were measured  at specific periods of time and the total period was noted.

The catheterization of the external jugular vein was performed with an epidural catheter of  0.9 mm internal diameter and external of 1.5 mm and introduced down 3 to 3.5 cm according to with the animal weight. The external connection to the infusion pump was with a Steffens model (Alice  King Chatham, Los Angeles CA and KDS 100, KDS Scientific Syringe Pumps Inc Boston, MA). The infusion liquid introduced was a Ringer lactate solution with 0.1 % bovine albumin at 0.25 ml/100 gr. weight/ hour for both groups; the catheter was controlled daily. The drug and the placebo were administered as in previous biochemical  studies.

All samples were frozen after centrifugation to –80ºC and analized using the same technique and reactives

4.Survival studies without parenteral hydration. ( 20 rats). It included three stages: Stage 1: Animals were housed  for a week; Stage 2: A surgical interventions similar to previous studies was performed; Stage 3: The survival period until the animals death. No hydration nor control of inorganic phosphorus and urea were considered in this study. Only the survival time until death ( cardio-respiratory arrest) was accounted.

            All the rats were anaesthetised using Uretane 1.000 mg/kg weight intraperitoneally every 6-8 hours. These studies followed the European regulations for research purposes.

            The statistical analysis was different for each case. In the biochemical study, the analysis of variance was used for quantitative data,  and the Fisher test for the statistical significance; the crossed diagrams  for the qualitative data and the student T test for the statistical differences of histologic score. The Kaplan  and Meier curves for the survival studies and the Log Rank test for the Statistical significance. The urea and the inorganic phosphorus rates were studied at two levels: 1. The variation on serum quantitative data was observed by analyzing the urea under the curve ( trapeze&rsquo;s method) until the first death occurred; 2. The variation on the velocity  (line&rsquo;s slope) of serum changes by fitted equations to minimal models; in both cases the analysis of variance and the Fisher test were used in order to find statistically significant differences (p<0.01).

RESULTS

            TABLE 2 shows the statistics results of the chemical study of the guts. The intestinal concentration of Na, K and Cl was lower in the treatment animals. The Na, K, Cl total content was lower in the treatment group  than the control. The intestinal concentration and total content of bicarbonate was higher in the treatment group than the control. The histological and ultrastructural microfilms of 4 intestinal speciments are shown in FIGURES 1,2 . The histologic score results were: 17 (2.46) for treatment group and 22.8 ( 2.46) for control group. The Student&rsquo;s T test was statistically significant (p <0.0001). Then, animals treated with SMS 201-995 had less lesions in their intestines respect to control animals.

            The survival studies results are shown in FIGURES 3,4,5,6. The mean survival level was 197.0 hours for the  hydrated treatment animals; 147.3 hours for the non-hydrated treatment animals; 55.6 hours for the hydrated non-pharmacotreated animals; and 41.9 hour for the non-hydrated, non-octreotide treated animals. Significant differences were obtained in all groups. The Kaplan and Meier  survival curves for the four groups of animals  with and without parenteral hydration are shown in FIGURES 3,4.

            The statistics results of the studies with BUN and inorganic phosphorus are presented in TABLE 3. The analysis of variance indicates that until the first animal death in both groups, the total amount of BUN is higher in the control than in the treatment group. Also, the amount of phosphorus produced is higher in the control group. The BUN and the inorganic phosphorus variation speed, represented by the curve gradients, were also faster in the control group. FIGURE 5,6. All the differences were statistically significant.

DISCUSSION

            This study gives more information about the SMS 201-995 and its effects on the guts in pathological situations.

            According to the biopharmacological base in which the octreotide uses the same mechanisms as the biological somatostatin we have elaborated a pathological pattern observed frequently in daily practice, the ischemia-revascularization syndrome; the intestinal ischemias  are  generally non-obstructive; the mesenteric  hipoperfusion is the cause of intestinal ischemia in 10% of cases; the  hipovolemic shock and some surgical techniques like transplants and aortic surgery  also produce intestinal ischemia (12,13,14). It has been observed that an ischemia of the guts lasting 90 to 120 minutes produces sufficient damage to the viability of the affected gut (15).  Moreover any temporary intestinal ischemia induces changes in the gut like disruption of the intestinal barrier and the subsequent alteration of the bacterial flora producing systemic effects like sepsis (16).

            In our investigation the basal electrolytic values of the experimental animals with the same breed and weight where within the normal limits, so no animal was excluded from the research. Therefore all differences observed in the treatment or control animals were due to the effect of SMS 201-995. Such differences in treatment animals are outlined:

The intestinal absorption of Na, K and water are maintained.

The chloride secretion and intraluminal bicarbonate content are maintained.

The four electrolitic serum concentration investigated are closed to the basal values.

The externa aspect of the intestinal villi is maintained. A minor lesion of the villi, the inflammatory infiltrate and the oedema of the intestinal layers.

The cytoprotective effect according to the rates of the intestinal phosphorus, where the cellular ischemic lesion is of less degree and appears later.

According to the speed of production of BUN, the renal plasma flow subsequently the plasma volume may have improved.

Alternatively the results obtained  on the animal control are similar to the ischemia- revascularization form. So the pharmacological effects of the SMS 201-995 over this syndrome are evident. The octreotide effects over the nonintestinal cases of ischemia-revascularization have been reported by different authors (17,18).

The natural result of the ischemic grade is the end of the oxidative  phosphorilation, the decrease of the ATP and its function as the active transport of the ATP. The ATP depletion produce Ca which is accumulated on the internal surface of the plasmatic membrane, activating the ATPasa Ca depenent and subsequently increasing the ATP depletion. The acumulated Ca activates diverse phospholipases and proteases and produces disruption on the plasmatic membrane and activates the intracellular proteolisis.

The ischemia produces a release of substances like histamine, VIP and prostanoids and its effects increase the membrane permeability and the apperance of the third space in the guts and subsequently the hipovolemia; this increase of liquid produces intestinal distention and a decrease of the mucosa vascularization due to compression. The ischemia also produces lesions on the intestinal mucosa barrier as the pancreatic proteolisis enzymes are released (19). All these elements produce alterations in the intestinal barrier and changes in the bacterial flora and the absortion-secretion intestinal processes will be effected (20).

The hypoxia and the absorption-secretion alterations of Cl/Na and H⁺/bicarbonate will produce intestinal acidosis and therefore an increase of the bacterial flora as well as alterations of the intestinal hemosthasia (21).The increase of the bacterial flora maintains the intestinal lumen acidosis. The subsequent toxemia will alter the intraparietal plexus causing an ileus paraliticus and producing alterations in the absorption- secretions system (22,23). As a result of the cellular hypoxemia, the proteolitic enzymes are released and the bacterial toxemia will produce cellular lesions (23). As the intracellular ions K and the inorganic phosphorus are released, their serum rate increase (24). The hypovolemia, the decrease of the diuresis and the hypercatabolic effect of the stress will icrease the BUN rates.

The results obtained from the investigations on the SMS 201- 995 and their effects on the experimental animals are indicated in the changes on the physiopathologic algorythm as shown in FIGURE 7:

The release of VIP is inhibited (25)

The proteases pancreatic release in inhibited (26)

The effects of the neurohumoral changes of the absorption-secretion on the somatostatin receptors in the mienteric plexus, the submucosa and the neurointestinal conexions are maintained (unconcluted research).

A cytoprotective effect due to: a) the cytoprotective prostanoid release (27); b) the effects of the free oxygen radicals are diminished (28); c) the cellular proliferation of the crypts and an increase exchange on the intestinal cells due to  the octreotide (the increase of LTB4); d) a decrease on the plateled activity factor on the mucosa (9).

The blockage of Ca activity &ndash; by direct action or by modifying the cAMP and cGMP cycles (4).

Regulation of the inflammatory response (23,29).

The influence on the intestinal motility (30,31).

All these actions permit:

To minimize the vasoactive effects of the medicators.

The maintenance of the peristalsis.

The alteration of the acid-base balance to be reduced to a  minimum as a overgrowth of the bacterial flora (as shown in the microfilms).

To minimize the hydroelectrolitic imbalance (as shown in the serum and intestinal electrolytes rates).

During the evolution of the revascularization stage, the haemorrhange on the

Intestinal lumen is observed , the hematocrit level decreases and emboli are liberated with the risk of the animals death. It is believed nowadays that the main cellular damage takes place during this stage and is due mainly to the free oxygen radicals produced during the ischemic stage and released during the revascularization stage (32,33).The cellular membrane lesions will produce  alterations  in the intestinal absorption-secretory system. Experimental studies demonstrate the effects of the octreotide  on the xantino-oxidase, that is, the effects of this molecule could alter the response of the macrofagocyte on the lipopolysacarides, producing a decrease of H₂O₂   and TNF secretion (29).A cytoprotective effect has been attributed to this pharmacologic factor due to the release of the free oxygen radicals (28). In our studies the group of treatment animals persent better results than the control, that id to say, the synthetic hormone has a clear effect on the cellular lesion during the ischemia-revascularization: although this effect could be observed in other studies during the ischemic stage (16), there is no evidence  of the pharmacological effect during the revascularization stage.

            The survival model proposed in this study consider the surgical lesion performed as lethal, in other words, when the animal were left without completing the treatment , the death was inevitable. This survival stage was considered an important factor in the beneficial effects observed on the biochemic and clinic studies demonstrating that the survival period was prolonged. The study was divided in two parts: with and without parenteral hydration; four different effects with two treatments were taken into account: without hydration or octreotide, with hydration and octreotide, octreotide without hydration, and hydration without octreotide. It was observed that the pharmacological effects on the survival stage were positive both with or without hydration.

            The BUN, a factor which increased in the hypovolemic stage, proves a valuable parameter on the renal hypoperfusion phase and therefore  a good  indicator of the severity  of the hipovolemic stage (34). Also as a product of the proteic metabolism it indicates the degree of proteic catabolism. Low rates of this metabolism on the treatment animals indicate a better hydration and a better control of the proteic catabolism although the survival rate is prolonged (35). The variation speed of the BUN in the treatment animals is not lineal as it normally occurs in these pathology but takes place in two stages:

Fast increase as in the control group.

Decrease.

It in effect shows that the pharmacological effects observed within the first 36 hours during the biochemical-study ara maintained , improving the renal function due probably to an increased volemia in response to an increase of the absorption-secretion function of the guts.

            The inorganic phosphorus and the sulphates are the most important anions within the intracellular compartment. In this work the inorganic phosphorus is an indicator of the cellular necrosis, according to the high levels of this ion on the intestinal ischemic processes (23,26).It gives information about the cytoprotective  effect of the  octreotide as is shown in our studies. Significant differences have been observed in the phosphoremia  between the treatment  and the control group in the gap  of timet where all the animals  were alive: in this period, the treatment animals had lower levels of phosphoremia respect to control animals. This gap appears at the beginning of the revascularization stage where the toxines increase on the ischemic guts. The (line`s slope) of Pi presents the same curve in the treatment and control group so the evolution of the phosphoremia is similar in both groups but it appears later in the treatment group. Therefore the pharmacological effects last until the death of the animals. According to the previous studies  the form of the curve (third degree model) is as expected in this type of pathology (37).

            The octreotide treated animals, with or without hydration, present a better survival rates and this  could  be due to:

A good hydroelectrolyte balance for a persistence of the intestinal functions as the cellular integrity  is maintained.

The control of the inflammatory reaction  which otherwise leads to a sepsis as  it occurs in the necrohemorrhagic pancreatitis (38).

A decrease of the proteic catabolism and its side effects.

Therefore octreotide given during the revascularization stage as the only treatment in the ischemic-revascularization syndrome and for a 90 min period total ischemia  produces a decreases in the liquids retention, maintains the Na, K intestinal absorption-secretion processes and has a positive effect on the acid-basis intestinal balance. These pharmacological properties improve the serum concentration of the four ions in the guts which remain within the basal levels during the revascularization stage.

It also decreases the degree of intestinal lesions. In the long term the octreotide has a cytoprotective effect, maintains an acceptable level of volemia and decreases the proteic autodigestion during metabolic stress on the revascularization stage. All these effects enables the prolongation of cellular survival.

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TABLE 1

TABLE 2

Biochemic- clinic studies. Intestinal ions result &ndash; media (SD)- cencentration (mEq/L) and total content (mEq) and the intestinal volume (before and after centrifugation) in mL and their statistics differences in the treatment and control animals.
 

Concentration (mEq/L)	TREATED	CONTROL	Fisher’s Test Value	P value
Na	124.36 (2.98)	142.36 (1.82)	27. 70	<0.0001
K	7.36 (0.58)	17.49 (1.28)	294.14	<0.0001
Cl	105.31 (4.06)	114.28 (2.76)	8.37	<0.0046
Bicarbonate	24.34 (2.15)	10.01 (1.32)	322.04	<0.0001
Intestinal content (mEq)	TREATED	CONTROL	Fisher&rsquo;s Test Value	P value
Na	0.40 (0.03)	1.03 (0.06)	57.09	<0.0001
K	0.023 (0.003)	0.125 (0.006)	178.26	<0.0001
Cl	0.341 (0.03)	0.830 (0.04)	38.24	<0.0001
Bicarbonate	0.127 (0.03)	0.067 (0.01)	54.42	<0.0001
Intestinal volumen (mL)	TREATED	CONTROL	Fisher&rsquo;s Test Value	P value
Before centrif	13. 25 (1.85)	17.35 (2.48)	16.09	<0.0001
After centrif	5.25 ( 1.25)	6.67 (1.04)	7.30	<0.008

TABLE 3

Survival study. The analysis of variance results with the inorganic phosphorus and BUN data: A. Quantitative grade of the variation (mean (SD)) and B. Velocity of variation  - curve&rsquo;s slope- (confidence intervals of the curves&rsquo; angular  coefficient). (The quantitative grade of the variation obtained according the area below de curve- until the first animal dead- according to the trapeze&rsquo;s rule; the velocity of variation  has been studied with equations fitted to minimum models and each section of the resultant curve has been compared- using the confidence intervals  of the angular coefficients of the curves- between treatment and control animal, to check if the sections were overlaped).

Legent: Beta 0 (curve value in the Y axis); Beta 1 ( time and section 1 of the curve); Beta 2 (time and section 2 of the curve); Beta 2 (time and section 2 of the curve); Beta 3 (time and section 3 of the curve).

A.

 

	AREA BUN	AREA Pi
TREATMENT	174.10 (3.37)	93.34 (5.34)
CONTROL	794.25 (30.80)	428.82 (45.50)
Fisher’s test value	488.11	536.22
P value	<0.0001	<0.0001

B.

 

	TREATMENT	CONTROL	P of no-overlaped
BUN
Beta 1	0.003602+/-0.000011	0.036344+/-0.004605	P<0.01
Beta 2	-0.0000169+/-0.00004	----	----
Pi
Beta 1	0.039939+/-0.003992	0.153388+/-0.019671	P<0.01
Beta 2	-0.000460+/-0.000046	-0.003841+/-0.000844	P<0.01
Beta 3	0.00000013+/-0.00000015	0.000325+/-0.0000096	P<0.01

FIGURES

A           B
FIG 1. Microfilms of a segment of ileum of the animals treated with octreotide (A) and the control (B). The ischemic lesion grade 1 (according to Chiu classification (11)) in the intestinal villi are evident in (A); the mucosal barrier is fairly intact; an acute inflamatoy infiltrate, oedema of the lamina propia and submucosa appears; mild vasodilation at all celular levels; no haemorrhage is observed. Ischemic lesions grade 3 are observed on the villi (B); the mucosal barrier is impaired and the axial sides of the villi are conserved; an important inflamatory infiltrate and oedema appear on the lamina propia and submucosa; moderate vasodilation at all levels (Hemtaxylin of Harris-eosine, x 20).

A          B   C         D
FIG 2.   Microfilms taken from a scanning electronic microscope of the ileum segment of one of the animals treated with octreotide (A,B), and the control grup (C, D). An opening of the villi apex, remarkable bacterial overgrowth, changes on the bacterial flora and red blood cell diapadesis are observed in C and D; therefore a celular substance from lamina propia is secreted through the open villi; the mucus secretion is absent. The external aspect of the villi shows a normal aspect and the mucus secetion is abundant, the bacterial overgrowth is moderate (A,B). (A and C, x 50; B and D, x 200).

FIG 3.   Kaplan and Meier curves on the treatment and control animals in the survival studies with parenteral hydration.

FIG 4. Kaplan and Meier curves on the treatment and control animals in the survival studies without parenteral hydration.

FIG 5. Curves according data fitted at minimun models; Velocity of variation (line's slope) of BUN in the treatment and control animals.

FIG. 6. Curves according data fitted at minimun models; Velocity of variation (line's slope) of Pi in the treatment and control animals.

FIG 7. Algorythm of physiopathologic events in the intestinal ischemia-revascularization. The frames shows the levels where the octreotide could have an effect.

Legend: Bic: bicarbonate; DAO: diaminoxydase; VIP: vasoactive intestinal peptide; PGF: prostaglandin F.