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The first species having circle of Willis (circulus arteriosus cerebri)?

The first species having circle of Willis (circulus arteriosus cerebri)?


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I know mammals are not the only species that have this anatomical unit (i.e. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982101/).

But I was wondering what was the first species that had a similar structure in their brain (or brain-like organ). Do we know this?


The Brain Willis Circle and Ring Electric Power Systems

Blood flowing to the brain keeps it alive, while electrons flowing to inhabited civilized places keep them active, leading to greater understanding of the world. What, however, of those many human beings still confined to distant hostile regions, unaware of the magic of electricity now over a century old … How do they live, what can they do, and what do we, happy people, do for them?

The word analogy is a synonym of likeness, resemblance, similitude, or affinity and involves two concepts being placed side by side, as in a comparison [1]. The workings of nature and those of human societies are amenable to such analogous comparison—even though the evolution of the natural world obviously spans millions of years [2], while human societies are much younger, relatively puppies by comparison. This article considers two interesting examples from these two realms that show remarkable similarities (possibly a result of sheer chance), i.e., a circulatory brain anastomosis, the circle of Willis (CW), and modern power transmission- distribution systems in the ring arrangement. Remember that electric networks handle the flow of charges [say, in coulombs per second (C/s) or electric charge per unit time, which is current), whereas hydraulic systems deal with fluid flow [say, in liters per minutes (L/min) or volume/unit time or fluid mass/unit time]. Hence, these systems too are analogous, a well-known fact often mentioned by instructors of electrical engineering courses.
Cerebral circulation refers to the movement of blood through the network of blood vessels supplying the brain, the primary governing organ that makes us human beings. The rate of cerebral blood flow in adults is typically 750 mm/min, representing about 15% of cardiac output [3]. The brain is very vulnerable to compromises in its blood supply consequently, its circulatory system has many safeguards, of which the CW is one. Brain circulatory failure results in cerebrovascular accidents, commonly known as strokes, a health situation of considerable concern.


The first species having circle of Willis (circulus arteriosus cerebri)? - Biology

Brain is one of the most metabolically active organs of the mammalian body receiving adequate blood supply, which is essential to maintain the glucose and oxygen levels. This demand is derived from circle of Willis (CoW) and its arterial anastomosis at the base of the brain.

To describe the arteries contributing to the formation of CW in sheep brain and to compare it with that of human. Sheep circle of Willis forms an animal model for undertaking further studies on blood supply of certain cranial nerves.

Materials and methods

In present study, 20 formalin fixed human brains from the Department of Anatomy, Mamata Medical College and 20 fresh sheep brains from local slaughterhouse were obtained. Sheep brain was removed as per the human brain removal procedure, and circle of Willis was studied by Radiological study.

Results

In sheep a well-developed four-sided arterial rete called ‘Rete Mirabile cerebri’ (RMC) was noted at the base of the brain in inter-peduncular fossa. Internal carotid arteries were either vestigial or absent in sheep. Two emergent arteries arising from steady pool of blood from RMC entered the inter-peduncular fossa. These arteries ascended on either side of infundibulum of pituitary gland and then diverged in cranial and caudal directions to form the Circle of Willis.


Anatomy of Circle of Willis

Also referred to as the Loop of Willis, the cerebral arterial circle or the Willis polygon, it is composed of five main arteries:

  1. Internal carotid artery (left and right)
  2. Anterior cerebral artery (left and right)
  3. Anterior communicating artery
  4. Posterior cerebral artery (left and right)
  5. Posterior communicating artery (left and right)

The CoW encircles the pituitary stalk, optic tracts and basal hypothalamus. It must be noted that anatomically, the CoW is not the same in every individual it is found to have anomalies in nearly 50% of people (Source).

There are two circulatory branches to supply blood to the brain. The two branches of the dorsal aorta supplies blood to the brain and the spinal cord. They are the internal carotid arteries or the anterior circulation of the brain which supplies blood to the anterior parts of the brain, the cerebral hemispheres and structures of the diencephalon (like thalamus and hypothalamus). The vertebral arteries form the posterior circulation and supply blood to the cerebellum, the brainstem and pons, as well as the posterior forebrain.

These two branches of cerebral circulation come together at the Circle of Willis.

The basilar artery, which forms one branch of the posterior cerebral circulation, joins the internal carotid arteries, the anterior cerebral circulation through the Circle of Willis. The two posterior communicating arteries and the anterior communicating artery are the three small bridging arteries. From these bridging arteries arise the posterior cerebral artery.

The anterior communicating arteries join the two anterior cerebral arteries (forming the anterior aspect of the CoW), whereas the posterior communicating arteries join the internal carotid arteries to the posterior cerebral arteries (forming the lateral aspect of the CoW).


Some observations on the cerebral arterial circles of mink (Mustela vison) †

Presented as a paper from the platform at the Eighty-first Session of the American Association of Anatomists, April 9–12, 1968. New Orleans, Louisiana. Anat. Rec., 160: 321 (abstract).

Abstract

With the exception of a brief allusion to an unidentified species of Mustela with regard to cerebral vascular studies by de Vriese ('05), major information concerning the circle of Willis in mink was nonexistent until the present investigation. Brains of mink in which the cerebral arterial circles were injected with latex and subsequently hardened in formalin, revealed the primary cerebral arterial anastomosis to be ring-like in form all of the component vessels were patent and well formed, none was attenuated or string-like.

Some of the more prominent findings included: (1) a predominance of asymmetric divergence of the posterior communicating arteries separating from the bifurcating basilar artery (2) the presence of a posterior intercommunicating artery in all of the specimens (3) the occasional doubling of the superior cerebellar and the posterior cerebral arteries (4) deeply placed internal cerebral loops forming secondary arterial anastomoses between some penetrating vessels in the caudal region of the circle other loops interconnected other penetrating vessels in the rostral region of the circle (5) blood channels forming an intercarotid anastomosis traversed the pia mater (6) the presence of a penetrating artery adjunctive to the recurrent artery of Heubner (7) anastomoses between the internal and the external ophthalmic arteries, and between the internal and the external olfactory arteries forming collateral channels of communication between the intracranial and the extracranial circulations (8) the occasional presence of an anterior communicating artery supplementing the commonly occurring azygos anterior cerebral artery which continued as a single vessel throughout its extent (9) unjoined anterior cerebral arteries in one animal which was exceptional.


Protokol til isolering af Mouse Circle of Willis

Den cerebral arteriel cirkel (Circulus arteriosus cerebri) eller kreds af Willis (CoW) er en kredsløbssygdomme anastomose omkring optiske chiasma og hypothalamus, der leverer blod til hjernen og de omkringliggende strukturer. Det har været impliceret i flere cerebrovaskulære lidelser, herunder cerebral amyloid angiopati (CAA) associeret vaskulopatier, intrakraniel aterosklerose og intrakranielle aneurismer. Undersøgelser af de molekylære mekanismer bag disse sygdomme til identifikation af nye lægemiddelkandidater til forebyggelse kræver dyremodeller. Nogle af disse modeller kan være transgene, mens andre vil involvere isolering af det cerebro-vaskulære gebet, herunder CoW.The her beskrevne fremgangsmåde er velegnet til CoW isolering i nogen mus afstamning og har et stort potentiale for screening (ekspression af gener, proteinproduktion, posttranslationelle protein modifikationer, secretome analyse, etc.) undersøgelser af de store skibe med musen cerebralekar. Den kan også anvendes til ex vivo undersøgelser, ved at tilpasse organbadet system udviklet for isolerede mus olfaktoriske arterier.

Introduction

Den cerebral arteriel cirkel (Circulus arteriosus cerebri), også kendt som kredsen af ​​Willis (CoW), løkke af Willisor Willis polygon) blev først beskrevet af Thomas Willis i 1664. Det er en kredsløbssygdomme anastomose placeret omkring den optiske chiasma og hypothalamus, der kan betragtes som et centralt knudepunkt tilfører blod til hjernen og omgivende strukturer. Blod ind denne struktur via den interne carotis og vertebrale arterier, og det strømmer ud af cirklen via den indvendige midten og posteriore cerebrale arterier. Hver af disse arterier har venstre og højre grene på hver side af cirklen. Basilar, post kommunikerer, og anterior kommunikerer arterier komplet cirklen (figur 1 og figur 2). Risikoen for nedsat blodgennemstrømning i nogen af ​​de udadgående arterier minimeres ved sammenlægning af blod ind i cirkel fra carotis og cerebrale arterier, hvilket sikrer, at tilstrækkelig blod tilføres til Bregn. Denne struktur fungerer også som den vigtigste rute for sikkerhedsstillelse blodgennemstrømningen i alvorlige okklusive sygdomme i det indre halspulsåre.

Flere typer af cerebrovaskulære sygdomme har deres oprindelse i koen. De mest almindelige er cerebral amyloid angiopati (CAA) associeret vaskulopatier, intrakraniel aterosklerose og intrakranielle aneurismer. 1, 2, 3 Disse lidelser kan føre til hypoperfusion grund vasodilation, og intracerebral og / eller subaraknoidal blødning i sidste ende omsætte til iskæmiske eller hæmoragisk slagtilfælde eller i bedste fald en forbigående iskæmisk anfald. Nylige fremskridt i diagnostiske procedurer, herunder Neuroimaging, eventuelt kombineret med angiografi, har gjort det muligt at diagnosticere disse store cerebrovaskulære sygdomme klinisk, uden behov for en hjerne biopsi. Ikke desto mindre er effektive og specifikke behandlinger (farmakologiske eller endovaskulære) øjeblikket mangler, og der er derfor behov for at definere nyemolekylære mål.

Identifikationen af ​​nye lægemiddelkandidater til forebyggelse af disse sygdomme hos mennesker vil kræve dyremodeller og måder at isolere cerebro-kar herunder koen. Sådanne modeller skal fremlægge dokumentation for og spor til de specifikke ændringer, herunder inflammatoriske forandringer, der forekommer i væggene i de store skibe i dyremodeller af intrakraniel arterie aneurisme, CAA eller intrakraniel åreforkalkning. 4, 5, 6

Vi har etableret en metode til mus CoW isolation for at lette undersøgelser af fartøj betændelse i Alzheimers sygdom (AD) og relaterede sygdomme, såsom CAA. Denne metode til isolering af muse koen blev udviklet til vurdering af inflammatoriske cerebrovaskulær genekspression under sygdomsprogression. Sammen med påvisningen af ​​amyloid beta deponering inden væggene af leptomeningeal og pial arterier, kunne denne metode gør det lettere at afskrækkemine det mulige forhold mellem inflammatorisk genekspression i cerebro-kar væg og Ap-peptid akkumulation. Det vaskulære netværk i hjernen, herunder leptomeningeal og pial i subarachnoidealrummet, er en udvidelse af de store arterier danner kredsen af ​​Willis. Den her beskrevne metode kan anvendes til at isolere CoW enhver muse afstamning og kan anvendes til alle typer af screening (fx genekspression, proteinproduktion og posttranslationelle protein modifikationer) på de store fartøjer af muse cerebro-vaskulære gebet.

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Protocol

Alle procedurer blev udført i overensstemmelse med EF-standarder for pasning og anvendelse af forsøgsdyr, med godkendelse af den lokale etiske komité for dyreforsøg (Ile de France-Paris-udvalget, Authorization 4270).

  1. Indgyde en dødelig dosis af pentobarbital (op til 1 mg / 10 g legemsvægt) intraperitonealt (27-gauge nål og 1 ml sprøjte) i voksne mus før operation.

BEMÆRK: Der er ingen grund til at anvende dyrlæge salve til øjnene under fartøj perfusion. Denne procedure er hurtig (5-10 minutter), og slutter i dyrets død. Bekræft manglende reaktion med en tå knivspids.

  1. Brug af iris saks, lave et snit, omkring 4 cm lange, i maveregionen og bughinden, lige under brystkassen.
  2. Lave et lille snit (nogle få millimeter dyb) i mellemgulvet og derefter continue snittet af membranen langs hele længden af ​​ribben bur for at blotlægge pleurahulen.
  3. Løft brystbenet væk og klemme spidsen af ​​brystbenet med arterieklemmen placere arterieklemmen på halsen. trim omhyggeligt fedtvæv forbinder brystbenet til hjertet.
  4. Passere 15-gauge perfusion nål gennem den venstre ventrikel ind i hjertespidsen.
  5. Endelig kan du bruge en saks til at klippe en af ​​leveren lapper til at oprette en stikkontakt.
    BEMÆRK: En alternativ afsætningsmulighed kan skabes ved hjælp af iris saks til at skabe et snit til højre atrium.
  6. Perfundere dyret med 25 til 50 ml phosphatbufret saltvand (PBS) med en pumpe drives ved en hastighed på 2,5 ml / min. Leveren skal blanchere som blodet er erstattet med PBS.
  7. Efter ca. fem minutter, når fluidet fra leveren er fuldstændig klar, stoppe perfusionen.
  8. Hvis der er planlagt immunofarvning eller almindelig farvning, perfundere dyret med 50 ml paraformaldehyd(PFA 4% i PBS) i 15 min.
    BEMÆRK: Forsigtig, PFA dampe er giftige. Perfusion af dyret med PFA bør udføres i et ventileret stinkskab.

3. Isolering af hjernen og Circle of Willis

  1. Isolering af hjernen
    1. Fjern hovedet med et par kirurgiske sakse.
    2. Foretag en midtlinjeincision med iris saks, langs huden fra halsen til næsen.
    3. Skær huden for at eksponere kraniet og fjerne eventuelle resterende muskler og fedtvæv med iris saks.
    4. Anbring den skarpe ende af iris saks ind i foramen magnum på den ene side og omhyggeligt skub dem langs den indre overflade af kraniet til den ydre øregang (også kendt som øregangen).
    5. Reproducere snittet beskrevet i 3.1.4 på den kontralaterale side og gøre en midtlinje skåret langs den indre overflade af den inter-parietal knogle til starten af ​​den sagittale sutur.
    6. Plant irissaks i den frontale knogler, lige mellem øjnene, i det sagittale sutur og derefter åbne dem at opdele kraniet i to.
    7. Løft hjernen, opsigtsvækkende olfaktoriske pærer og bruge iris saks at afskære nerve forbindelser på dens ventrale overflade.
    8. Fjern hjernen og læg den i en 60-mm petriskål indeholdende iskold PBS for rå isolation. Helt fordybe hjernen i PBS. Hvis hjernen blev fikseret med 4% PFA (til efterfølgende sektionering og immunfarvning eller almindelig farvning), holde det i et bad af 4% PFA ved 4 ° C i 24 timer.
    1. Sæt hjernen på hovedet (dvs. på dens dorsale overflade) til at visualisere koen.
    2. Brug en lille pincet til at få fat i de forreste cerebrale arterier (ACA) i bunden af ​​de olfaktoriske lapper ( 7.


    Figur 1: Skematisk diagram af en ventrale View af Mouse Brain Fremhævning koen koen er dannet af de to interne halspulsårer (MCA), som er afledt af de to forreste cerebrale arterier (ACA). basilararterie (BA) filialer i den bageste (PCA) og overlegen (SCA) cerebrale arterier, og to vertebrale arterier (VA).

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    Representative Results

    PBS-perfunderet mus bliver dræbt, og koen er isoleret som beskrevet i afsnit 3.2 i protokollen. Når dissektion udføres korrekt, bør koen kommer ud i et stykke og skal være lidt gennemsigtig på grund af fraværet af tilbageværende blod i karrene.


    Figur 2: Mus CoW efter Isolation. (A) Oversigt over ko i en 10 cm petriskål. (B) Nærmere oplysninger om de forskellige grene af koen. MCA for mellemledere cerebrale arterier, ACA for anteriore cerebrale arterier, BA for basilar arterie, PCA for bageste cerebrale arterier og SCA for overlegne cerebrale arterier. Klik her for at se en større version af dette tal.

    telt. "fo: holde-together.within-side =" 1 "> Renheden af koen præparat kan kontrolleres ved at sikre, at specifikke vaskulære gener stærkt udtrykkes mens ekspressionen af neuronale gener er målbart Mere specifikt bør det rensede CoW specifikt udtrykker vaskulær glat muskulatur cellemarkører, såsom glatmuskelactin (SMA), glat muskulatur-myosin tung kæde (SM-MHC) og smoothelin. Disse markører er næppe udtrykkes i andre dele af hjernen. en modsat ekspressionsmønster bør være opnået for neuronale gener, med neuronale markører (MOG, MAP2) kun knap påviselige i koen.

    Niveauer af mRNA kan vurderes ved RT-qPCR med normalisering i forhold til en reference gentranskript (her, den for GAPDH). Typiske resultater er vist i figur 3A og B.

    Figur 3:. Expression af neuronal og kontraktile gener i mus Circle of Willis og Brain De RT-qPCR resultater blev normaliseret mod dem for en reference gen (GAPDH). De er udtrykt som middelværdien ± SD af 6 til 12 uafhængige forsøg Sammenligning af ko med hjernen: ns, ikke signifikant, *, P <0,05, ** P <0,005, og ***, P <0,001 (A) Ekspression af kontraktile gener:. SMA (glatmuskelactin), smoothelin, SM-MHC (glat muskulatur-myosin tung kæde 11) og E-selectin (B) Ekspression af neuronale gener:. Mog (myelin oligodendrocyt glycoprotein) og Map2 (mikrotubulus-associeret protein 2).

    Ekspressionsmønsteret for den endotel markør E-selectin i hjernen mangler Koen er meget lig den, der opnås for rå prøver, hvilket muligvis afspejler eksistensen af ​​en hjerne kapillær netværk. Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    Vi beskriver her en reproducerbar protokol til isolering af kredsen af ​​Willis. De mest almindelige cerebrovaskulære lidelser, der involverer koen er CAA-associerede vasculopatier, intrakraniel åreforkalkning og intrakranielt aneurisme, som alle påvirker væggene i blodkar. Risikofaktorerne er velkendte, men den molekylære patogenese af disse cerebrale lidelser stadig dårligt forstået og specifikke biologiske markører til at forudsige deres forekomst mangler. Der er stor interesse for metoder til isolering koen fra transgene mus til at linke makroskopiske observationer med molekylære ændringer. For eksempel ved at inducere konsistente aneurismer i en musemodel, hvori et bestemt gen slås ud (som beskrevet af Hosaka et al. 8) og analysere ko, bør det være muligt at bestemme, om behandlinger rettet mod proteinet kodet af dette gen kunne forebygge intrakranielle aneurismer og / eller subaraknoidal blødning. Obviously kunne denne fremgangsmåde også anvendes til at analysere effekten af ​​et bestemt lægemiddel på progression af CAA-associeret vasculopatier i de forskellige transgene musemodeller for AD.

    Det er lettere at isolere muse arterier end at rense hele muse mikrokar, men sådan prøveudtagning er imidlertid vanskeligere i mus end i rotter. Faktisk er rotte CoW indlejret i meget stivere meninges, hvilket gør det muligt at isolere hele strukturen på én gang. Ud over problemet med den lille størrelse af fartøjer i mus, denne procedure er også vanskelig på grund af gennemsigtigheden af ​​de fartøjer, efter deres perfusion med PBS før fjernelsen af ​​hjernen. Vi anbefaler derfor at praktisere uden infusion, begyndende fra trin 3 efter anæstesi for at gøre det lettere at skelne blodkarrene. Endelig, som mus cerebrale kar er meget fint og knækker let, at dissektion skal udføres meget omhyggeligt, uden farende, for at sikre, at hele strukturenisoleres i et stykke, ved først at tage fat i ACA. Renheden af ​​CoW præparat kan evalueres ved at sammenligne niveauerne af ekspression af de neuronale og fartøjets gener. Med praksis, en enkelt CoW giver kun tilstrækkelig RNA for studiet af 5 - 10 genekspressioner. For storstilet screening bør flere mus køer samles.

    Gauthier et al. 9 beskrevet en fremgangsmåde til isolering små stykker af mikrokar (baseret på anvendelsen af collagenase / dispase at fordøje fragmenter af hjernen og af glasperler til fælde dem), som giver glatte muskulatur eller endotelceller, der kan opretholdes i kultur, når anbringes i et passende medium. Fordelen ved vores metode er, at den isolerer store skibe, uden at ændre fartøjets struktur. Dissektion af specifikke grene af koen kan også være af interesse, for at bestemme korrelationen mellem genekspressionsprofilen af ​​en bestemt gren og eventuel modtagelighed for cerebrovaskulære diseases, såsom hjerne-aneurisme. Sådanne tilgange kan give en forklaring på, hvorfor nogle ACA anatomiske og genetiske variationer er korreleret med en højere forekomst af ACA aneurismer. Denne dissektion kræver tryk, der skal udøves med pincet ved bunden af ​​hver specifik gren, at dissociere det fra de andre grene, ved starten af ​​koen isolation procedure.

    Således, ud over at gøre det muligt at udlede celler fra koen 10 eller til at screene for genekspression 11 og proteinproduktion 12 eller posttranslationelle protein modifikationer, denne metode kan anvendes til ex vivo-undersøgelser, ved blot at tilpasse organbadet systemet udviklet til isoleret muse olfaktoriske arterier. 7 efter inkubering af hele ko i dyrkningsmedium indeholdende antibiotika, kan opnås og analyseres secretome frigivet af denne specifikke struktur. En analyse af denne secretome i musemodeller for CAA ville gøre det possible, for eksempel for at bestemme CAA-relaterede inflammatorisk status for disse cerebrale arterier. Det resulterende konditionerede medium kunne også anvendes til at bestemme virkningerne af den secretome på hver karvæggen celletype fænotype. Patologiske COW præparater kunne også anvendes til at identificere eventuelle forskelle i biokemiske signaler, såsom cyklisk guanosinmonophosphat (cGMP), cyklisk adenosinmonophosphat (cAMP) eller Ca2 + koncentrationer detekterbare med fluorescensresonansenergioverførsel (FRET) -baserede biosensorer. Virus-leveret biosensorer er blevet brugt på hjernens slice præparater indeholdende eksperimentelt tilgængelige modne levende neuroner med et bevaret morfologi, hvilket fører til påvisning af afgørende forskel D1 respons mellem de pyramideformede kortikale neuroner og striatale medium piggede neuroner. 13

    Modelleringen af cerebrovaskulære sygdomme i transgene mus, der udtrykker FRET-baserede anden messenger sensor proteiner 14 til billeddannelse af biokemiske signaler i den isolerede koen skulle yderligere give yderligere indsigt i biokemi, patofysiologi, og farmakoterapi af disse sygdomme.

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    Acknowledgments

    Dette arbejde blev støttet af Paris VI University og en Pierre Fabre Innovation tilskud.


    Anesthesia

    The complexity of surgical procedures required to induce cerebral ischemia in large animal species requires anesthesia, often for prolonged periods of time. Surgical preparation in TBI models is typically much shorter nevertheless, anesthesia needs to be adequately planned with suitable monitoring throughout. Although anesthetic agents are often administered during neurosurgical procedures, it must be recognized that human patients are rarely under their influence when suffering a stroke or TBI, and this must be acknowledged as a potential variable. Despite this, principles that apply to clinical anesthesia are recognized as appropriate for large animal models. These conditions best preserve normal neurological function throughout and thereby facilitate the accurate determination of deficits applicable to the induced injury.

    Preanesthetic considerations and induction.

    It is recommended that all large animals requiring long-duration anesthesia be fasted before surgery to reduce the risk of intraoperative regurgitation. Veterinary practice recommends food be withheld for a minimum of 12 h in feline, canine, porcine, and NHP species (86). There is some speculation regarding optimum fasting duration in ruminant species, as fasting has been shown to have little effect on ruminal contents (153). Fasting may however benefit a reduction in ruminal tympany, in which gas accumulates in the rumen due to the bacterial fermentation process. It is therefore advised food and water be withheld for 6 h to reduce gaseous accretion.

    For the purpose of all large animal surgery, it is highly recommended animals be induced with a sedative to ease handling, provide restraint, reduce distress, and in turn decrease the required dose rate for maintenance anesthesia by 30–50% (69). Commonly reported induction agents and associated dosing regimes are detailed in Table 1.

    Table 1. Recommended preanesthetic treatment regimens for feline, canine, porcine, ovine, and nonhuman primate species

    Scheme: dose rate, route of administration (per os, iv, sc, im), effect, and notes. NHP, nonhuman primates. Table adapted from Laboratory Animal Anesthesia (Fourth Edition), by Flecknell PA (71a), Chapter 5-Anaesthesia of Common Laboratory Species: Special Considerations, p. 227, 233, 239, 243, 247, copyright 2016 with permission from Elsevier.

    Anesthetic induction and sedation also allow for ease of intubation and endotracheal tube insertion (88). Intubation of the animal allows control of ventilation by mechanical means, thus facilitating regulation of the respiratory component of both pH and P co 2. Although spontaneous breathing is reported, it is often accompanied by hypoventilation and consequent increase in lesion volume (111). Controlled ventilation in large animals offers significant advantage over rodents and other small animals, which can be difficult to intubate due to the small circumference of the larynx. Physical intubation in large animals is fairly uniform across species. However, the laryngeal anatomy of porcine species is unique due to the situation of the larynx within an airway sigmoid curve, which may lead to intubation difficulties (83). Consultation with an experienced veterinarian is therefore highly advised before endotracheal tube insertion in these species.

    It is recommended that mechanically ventilated animals be kept on a combination of air/oxygen or pure oxygen to both maintain blood oxygenation levels and meet metabolic demands. The latter is influenced by variables such as temperature, body weight, and anesthetic agent used, and thus oxygen delivery must be adjusted accordingly (156). Maintaining animals on pure oxygen for durations >12 h is not recommended, however, due to the potential development of pulmonary edema and oxygen toxicity (82). The mechanical rate of ventilation for each species must be considered in relation to normal respiration rate and body mass so as to best maintain physiological P o 2 and P co 2 levels (146). Species-specific physiological variables, including normal respiration rates and recommended ventilation, are summarized in Table 2 (70).

    Table 2. Summary of physiological variables of feline, canine, porcine, ovine, and nonhuman primate species

    Anesthetic maintenance.

    Following induction, intraoperative maintenance of anesthesia can be achieved through the use of both intravenous and inhalational agents. A comprehensive summary of commonly used intravenous, intramuscular, and subcutaneous anesthetic agents and recommended dosing regimes is detailed in Table 3 and inhalational agents in Table 4.

    Table 3. Recommended anesthetic dose regimes for feline, canine, porcine, ovine, and nonhuman primate species

    Duration of anesthesia is provided only as a general guide, since considerable between-animal variation occurs. Scheme: dose rate, route of administration (per os, iv, sc, im), effect, and notes. NHP, nonhuman primate. Table adapted from Laboratory Animal Anesthesia (Fourth Edition), by Flecknell PA (71a), Chapter 5-Anaesthesia of Common Laboratory Species: Special Considerations, p. 229, 234, 240, 245, 248, copyright 2016 with permission from Elsevier.

    Table 4. Recommended minimal alveolar concentration of commonly used inhalational anesthetic agents for use in feline, canine, porcine, ovine, and nonhuman primate species

    The use of intravenous anesthetic agents may be preferential for imaging purposes, where the use of equipment required for inhalational anesthesia may be unfeasible, such as high-field MRI. However, inhalational agents are typically used, especially for longer duration procedures. Inhalational anesthetics agents used in rodent and large animal studies tend to be similar, with preference for isoflurane commonly reported in canine, NHP, ovine, and porcine models (34, 150, 168, 193). However, respiratory depression and hypotension associated with isoflurane administration (96, 118) contraindicate prolonged surgical use, requiring close monitoring of respiratory and blood pressure (BP) parameters, in conjunction with anesthesia depth. Maintaining animals on a level of isoflurane that maintains the minimum alveolar content <1.5 is highly recommended as it reduces the likelihood of adverse events (9). Intravenous and inhalational anesthetics may thus be used in combination to eliminate the likelihood of adverse events if an appropriate depth of anesthesia is not achievable under 1.5 minimum alveolar content isoflurane alone. This can be achieved through continuous intravenous infusion using a pump and adjusting dose rates to achieve the desired level of anesthesia. Intravenous anesthetic agents such as propofol and ketamine have been used in NHP, porcine, and ovine studies in conjunction with inhalational anesthetics with reported success (9, 46, 98, 118, 193). Recommended mean alveolar concentrations of various inhalational anesthetic agents are detailed in Table 4 and can be used in conjunction with dosing regimes from Table 4 (21).

    Given the aforementioned length of anesthesia required for many large animal models, the potential for neuroprotection brought about by anesthetic regime may contraindicate use of certain agents for extended durations. Valid animal modeling of acute CNS injury requires minimal interference from anesthetic and analgetic agents to best preserve the natural course of brain injury, while minimizing animal suffering (162). Agents such as ketamine exhibit neuroprotective properties via inhibition of the N-methyl- d -aspartate receptor (96, 142), which has been shown to attenuate the deleterious neurochemical sequelae following brain injury (130). However, ketamine use has also been shown to increase cerebral oxygen metabolic rate, which has implications for lesion volume and number of neuronal cells affected (31). Maintenance of animals on a combination of ketamine and isoflurane may offer a beneficial alternative in surgical procedures requiring prolonged duration (>4 h) anesthesia and has been reported in porcine, ovine, and NHP models (150, 168, 192). The anesthetic combination produces a countering effect, eliminating the neuroprotective effect of pure ketamine and the respiratory distress associated with pure isoflurane, thus reducing confounding factors of anesthetic agents administered alone (192, 193).

    In addition to potential confounding effects of anesthesia on outcome parameters, there are several species-specific complications that require consideration before study commencement. Pigs are especially prone to intraoperative development of malignant hypotension and malignant hyperthermia, both of which can have fatal consequences (36, 119, 191). Perianesthetic mortality is also common in feline and canine species, 100 times that of humans, and thus the animal must be carefully monitored for depth of anesthesia (27). As a result of the fermentation process in ruminating species, surgical positioning must be carefully considered to avoid excessive pressure on the rumen as up to 25% of anesthetized animals will regurgitate intraoperatively (153). Irrespective of preoperative fasting to reduce ruminal tympany, in some cases appropriate degassing should be performed to avoid excessive pressure on the diaphragm, which can limit ventilation (26). Ruminants salivate excessively, and for procedures requiring extended duration anesthesia, it is highly recommended that the alkaline saliva is collected and returned to the animal via an orogastric tube to prevent the development of acidosis (153). For other species, such as the cat and dog, pretreatment with an anticholinergic agent such as atropine can significantly reduce intraoperative salivation (Table 1).

    Evidence calls for rigorous planning of anesthetic regimes before study commencement, taking into consideration the potential effects on depth and duration of anesthesia, adverse events, and potential confounding effects of anesthesia on secondary injury processes.


    Analysis of morphological variation of the internal ophthalmic artery in the chinchilla (Chinchilla laniger, Molina)

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    Three-dimensional hemodynamics analysis of the circle of Willis in the patient-specific nonintegral arterial structures

    The hemodynamic alteration in the cerebral circulation caused by the geometric variations in the cerebral circulation arterial network of the circle of Wills (CoW) can lead to fatal ischemic attacks in the brain. The geometric variations due to impairment in the arterial network result in incomplete cerebral arterial structure of CoW and inadequate blood supply to the brain. Therefore, it is of great importance to understand the hemodynamics of the CoW, for efficiently and precisely evaluating the status of blood supply to the brain. In this paper, three-dimensional computational fluid dynamics of the main CoW vasculature coupled with zero-dimensional lumped parameter model boundary condition for the CoW outflow boundaries is developed for analysis of the blood flow distribution in the incomplete CoW cerebral arterial structures. The geometric models in our study cover the arterial segments from the aorta to the cerebral arteries, which can allow us to take into account the innate patient-specific resistance of the arterial trees. Numerical simulations of the governing fluid mechanics are performed to determine the CoW arterial structural hemodynamics, for illustrating the redistribution of the blood flow in CoW due to the structural variations. We have evaluated our coupling methodology in five patient-specific cases that were diagnosed with the absence of efferent vessels or impairment in the connective arteries in their CoWs. The velocity profiles calculated by our approach in the segments of the patient-specific arterial structures are found to be very close to the Doppler ultrasound measurements. The accuracy and consistency of our hemodynamic results have been improved (to (16.1 pm 18.5) %) compared to that of the pure-resistance boundary conditions (of 43.5 (pm ) 28 %). Based on our grouping of the five cases according to the occurrence of unilateral occlusion in vertebral arteries, the inter-comparison has shown that (i) the flow reduction in posterior cerebral arteries is the consequence of the unilateral vertebral arterial occlusion, and (ii) the flow rate in the anterior cerebral arteries is correlated with the posterior structural variations. This study shows that our coupling approach is capable of providing comprehensive information of the hemodynamic alterations in the pathological CoW arterial structures. The information generated by our methodology can enable evaluation of both the functional and structural status of the clinically significant symptoms, for assisting the treatment decision-making.

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    Sharmila P Bhanu 1 , Suneetha Pentyala 2 , Devi K Sankar 1

    1 Department of of Anatomy, Narayana Medical College, Nellore, Andhra Pradesh, 2 Department of of Radiology, Narayana Medical College & General Hospital, Nellore, Andhra Pradesh, India

    Correspondence to:Devi K Sankar
    Department of of Anatomy, Narayana Medical College, Chinthareddypalem, Nellore, Andhra Pradesh 524003, India
    E-mail: [email protected]

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    The posterior communicating arteries (PCoA) are important component of collateral circulation between the anterior and posterior part of circle of Willis (CW). The hypoplasia or aplasia of PCoA will reflect on prognosis of the neurological diseases. Precise studies of the incidence of hypoplastic PCoA in Andhra Pradesh population of India are hitherto unreported, since the present study was undertaken. Two hundred and thirty one magnetic resonance angiography (MRA) images were analyzed to identify the hypoplasia of PCoA and presence of fetal type of posterior cerebral artery (f-PCA) in patients with different neurological symptoms. All the patients underwent 3.0T MRI exposure. The results were statistically analysed. A total of 63 (27.3%) PCoA hypoplasia and 13 cases with f-PCA (5.6%) cases were identified. The hypoplastic PCoA was noted more in males than females (P<0.05) and right side hypoplasia was common than the left (P<0.04) bilateral hypoplasia of PCoA was seen in 37 cases out of 63 and is significant. The hypoplastic cases of the present study also were associated with variations of anterior cerebral arteries and one case was having vertebral artery hypoplasia. Incidence of PCoA as unilateral or bilateral with other associated anomalies of CW is more prone to develop stroke, migraine and cognitive dysfunction. Knowledge of these variations in the PCoA plays a pivotal role in diagnoses of neurological disorders and in neurovascular surgeries and angiographic point of view.

    Keywords: Posterior communicating artery, Hypoplastic, Posterior cerebral artery, Circle of Willis, Cerebral artery

    Introduction

    Detailed anatomy of the circle of Willis (CW) or circulus arteriosus is important in the field of neurology, neurosurgery and anatomy. The CW is the main arterial structure located at base of the brain that establishes collateral circulation to the brain and surrounding structures. The arterial circle is formed by internal carotid and vertebro-basilar systems comprising anterior cerebral artery (ACA), proximal segments of internal carotid and proximal segments of posterior cerebral arteries from basilar artery. The CW normally equalizes the blood flow to various parts of the brain. But a complete CW is seen in minority of the population and as age advances it shows different types of anatomical variations in its branches since birth [ 1 ].

    Posterior communicating artery (PCoA), branch of internal carotid artery acts as a significant anastomotic channel between anterior and posterior cerebral circulations [ 2 ]. Each PCoA runs postero-medially and anastomose at the junction between pre (P1) and post-communicating (P2) segments of ipsilateral posterior cerebral artery (PCA) [ 3 ]. The PCoA occasionally continues as PCA, called as fetal PCA (f-PCA) with a complete absence of P1 segment [ 4 ]. Its occurrence may be unilateral or bilateral, and in these conditions, the PCoA is bigger than the normal [ 5 ].

    The PCoA establishes the collateral circulation through its penetrating branches which supply the ventrolateral and dorsomedial thalamic nuclei, tuber cinereum, mamillary bodies, and cerebral peduncles [ 6 ]. The PCoA hypoplasia and aplasia can be congenital variations characterized by a narrow or poorly developed artery with limited blood flow [ 1 ]. As PCoA render a vital communication between internal carotid and vertebro-basilar system, occlusion, aplasia or hypoplasia of this artery can significantly affect the vascularity of brain. In bilateral aplasia or hypoplasia, there will be a bilateral interruption of blood supply to the cerebellum. These anatomical variations reduce the accessibility of collateral vessels and its circulation. Hence identification of such variations is important in the evaluation of cerebral vascular morbidity and its allied treatments. The objective of the present study is to observe the variations in the arrangement of PCoA of CW.

    Materials and Methods

    The present study was a retrospective analysis of magnetic resonance angiography (MRA) of CW on 231 patients, which included 154 males and 77 females. The age of the patients with hypoplastic PCoA was minimum of 25 years and maximum of 79 years. The study was approved by the institutional ethical committee and clinical variables were abstracted from Institutional Neurology review board. The cases were obtained between the years 2016&ndash2019 from the patients who had evidence of cerebral ischemic stroke (CIS), history of severe migraine, less hearing sense, dim vision and mild focal neurological deficit.

    A time of flight (TOF)-MRA technique was used and the study was conducted and analyzed in the departments of radiology, anatomy, and neurology. All the patients underwent 3D TOF-MRA using 3.0T MRI machine (3.0T system, GE Discovery MR750w 3.0Tesla GE Healthcare, Milwaukee, WI, USA) and imaging parameters used were (1) repetition time was 30 milliseconds and echo time was 2.7&ndash3.1 milliseconds, (2) flip angle was 20°, (3) 200 mm field of view, (4) section thickness was 1.4 mm, and (5) the imaging time was approximately 4.49 minutes. The images obtained were processed using a maximum intensity projection algorithm to create an angiogram like image [ 7 ]. The reconstructed images were then analyzed to detect the hypoplastic (<1 mm in diameter) or aplastic PCoA and with or without the f-PCA. The PCoA with aneurysms were excluded from the study. Other than hypoplasia of PCoA, anomalies such as hypoplasia or aplasia of other cerebral arteries and its branches were also noted. The data obtained were analyzed with Statistical Package for Social Sciences software (IBM SPSS Statistics for Windows, Version 25.0. IBM Corp., Armonk, NY, USA). The statistical dependencies between age, side and sex were measured using the Student t-test. The differences between the male and female PCoA hypoplasia were assessed in relation to side using the Chi-square test. Probability values of P >0.05 were considered as statistically significant.

    Results

    In the present study, out of 231 patients, 63 patients (27.3%) showed the incidence of hypoplastic PCoA in MRA pictures, which included 39 males (16.9%) and 24 females (10.4%). The mean age of males and females were 65.56±8.18 and 56.83±12.22 respectively ( Table 1 ). The hypoplasia of PCoA was noted more in males than females ( P <0.05). Out of 63 PCOA hypoplastic, unilateral cases included 26 of which right side PCoA hypoplasia ( Fig. 1A ) in males was 9 and in females 7 while the left side ( Figs. 1B , 2A , 4B ) was found to be 6 in males and 4 in females. The right sided hypoplasia was significant ( P <0.04) than the left in the present study. The bilateral hypoplasia ( Figs. 2B , 3A , 3B , 4A ) was seen in 37 patients (58.7%), which included 24 males and 13 females and is found significant. Overall the statistical association between the hypoplasia of PCoA in relation to sex and side was found to be highly significant ( P <0.001) ( Table 2 ).

    Table 1 . Independent sample-T and ANOVA tests to find the differences between the sex and side in patients with hypoplastic posterior communicating artery

    Variable Male (in years) Female (in years) Totalt-value P -value
    SideNMean±SDMinMaxNMean±SDMinMaxNMean±SD
    Left side662.50±14.384579448.50±15.3529651056.90±15.671.4700.180a)
    Right side969.00±6.636074755.29±17.3025741663.00±13.882.1970.045b)
    Bilateral2465.04±6.0452791360.23±6.3952753763.35±6.812.1530.038b)
    Grand total3965.56±8.1845792456.83±12.2225756362.24±10.713.3990.001c)
    F-value0.9562.2811.507
    P -value0.393a)0.122a)0.230a)

    N, number of cases observed SD, standard deviation Min, minimum Max, maximum t, results of independent sample t-test P , difference between the sex, age and side & P <0.05 is considered significant F and P are the results of ANOVA. a) Not significant b) Significant c) Highly significant

    Table 2 . Cross-tabulation of side and sex-specific incidence of hypoplastic posterior communicating artery patients

    SideNMaleFemaleTotalChi-square value P -value
    Left sideCount6410
    % within left side60.0040.00100.00
    % within sex15.3816.6715.87
    Right sideCount9716
    % within left side56.2543.75100.00
    % within sex23.0829.1725.400.3690.831a)
    BilateralCount241337
    % within left side64.8635.14100.00
    % within sex61.5454.1758.73
    Grand totalCount392463
    % within left side61.9038.10100.00
    % within sex100.00100.00100.00

    N, number of cases observed. a) P -value by Chi-square test, not significant

    Figure 1. MRA images showing (A) hypoplasia of right side PCoA (arrowhead) with left fetal type of posterior cerebral artery (arrow) (B) left side PCoA hypoplasia (arrowhead) with hypoplastic left A1 segment of anterior cerebral artery (asterisk). MRA, magnetic resonance angiography PCoA, posterior communicating artery.
    Figure 2. MRA images showing (A) left side PCoA hypoplasia (arrowhead) (B) bilateral PCoA hypoplasia (arrowheads). MRA, magnetic resonance angiography PCoA, posterior communicating artery.
    Figure 3. MRA images showing (A) bilateral PCoA hypoplasia (arrowheads) (B) bilateral PCoA hypoplasia (arrowheads) with hypoplastic A1 segment of anterior cerebral artery (asterisk). MRA, magnetic resonance angiography PCoA, posterior communicating artery.
    Figure 4. MRA images showing (A) bilateral PCoA hypoplasia (arrowheads) with hypoplastic right vertebral artery (arrow) (B) left side PCoA hypoplasia (arrowhead) with right fetal type of posterior cerebral artery (arrow) this case also presented hypoplastic right A1 segment of anterior cerebral artery (asterisk) and hypoplastic right vertebral artery (double arrows). MRA, magnetic resonance angiography PCoA, posterior communicating artery.

    The f-PCA was observed in 13 (5.6%) cases which is more on right (73.0%) ( Fig. 4B ) than left side (62.6%) ( Fig. 1B ) and the incidence is more or less equal in both males and females with no statistical significance ( P <0.05) ( Table 3 ).

    Table 3 . Incidence of fetal PCA in relation to sex and side

    VariablePresence of fetal PCA (N=13)
    RightLeft P -value
    Male (n=154)3 (1.9%)3 (1.9%)1.0a)
    Female (n=77)4 (5.2%)3 (3.9%)0.7a)
    Chi-square value0.0660.7b)

    PCA, posterior cerebral artery N, number of fetal PCA cases observed P , difference between the sex and side & P <0.05 is considered significant n, total number of males or females. a) Not significant b) P -value by Chi-square test, not significant percentages are mentioned within brackets

    Apart from hypoplasia of PCoA and f-PCA, some of the cases also presented with other arterial anomalies. Out of 10 left PCoA hypoplastic cases, 2 cases were associated with hypoplastic right vertebral artery ( Fig. 4A, B ), right A1 segment of ACA and right f-PCA ( Fig. 4B ) and left A1 segment of ACA ( Fig. 1B ). Similarly on right PCoA hypoplasia, 6 out of 16 cases were identified with hypoplastic A1 segment of ACA. In total of 37 bilateral hypoplastic PCoA, 4 were associated with hypoplastic A1 segment of ACA ( Fig. 1B ) and one cases was found to be associated with vertebral artery hypoplasia ( Fig. 4A ).

    Discussion

    The CW is the main source of blood supply to major parts of the brain, in which various patterns of its formation and number of anatomical variations have been reported till date in the literature. In most variations of the CW, brain function may not be affected due to the collateral circulation and compensation of the blood supply from the contralateral side. In a study of 1,000 brain specimens, 45.2% of typical and 54.8% variations of the CW were reported [ 8 ].

    The PCoA is an important artery establishing collateral circulation between the anterior and posterior part of CW. This artery also acts as an exit port for thalamoperforating artery, in which the important branch is pre-mamillary or thalamotuberal artery [ 9 ] which supplies the floor of 3rd ventricle, thalamus, hypothalamus, mammillary bodies, tuber cinereum, optic tract, pituitary stalk, cerebral peduncle and posterior perforated substance [ 10 ]. According to a previous report, aplasia of the right PCoA (16.6%) is more common than the left (3.3%) which is in accordance with the present study [ 11 ]. PCoA hypoplasia was found to have a pathophysiological role in stroke with or without carotid artery occlusion [ 6 ]. Out of all the branches of CW, a single branch occlusion might not lead to ischemia, because the collateral supply of that particular region will take over the function [ 12 ]. However, PCoA hypoplasia would be prone for the ischemia since its perforating branches might be scarcely perfused in cases of PCoA hypoplasia, predisposing thalamic infarctions leading to lacunar stroke [ 13 ]. PCoA hypoplasia as an independent or in association with anterior communicating artery and vertebral artery as observed in the present study can be suggested as a risk factor for ischemic stroke [ 14 , 15 ].

    In a study of classical CW, 10% of aplasia, hypoplasia or double PCoA have been reported, stressing the importance of PCoA in vertebral artery hypoplasia which results in stroke [ 16 ]. However, in the present study double PCoA has not been observed. But one case of vertebral artery hypoplasia along with bilateral PCoA hypoplasia has been noticed. Vertebral artery hypoplasia or occlusion is rarely symptomatic because there will be a sufficient collateral circulation from the contralateral side through CW. But in conditions of bilateral PCoA and one of the vertebral artery hypoplasia, size and patency of these collateral pathways may be a risk factor for developing cerebral infarction [ 17 ].

    In the present study, 13 cases of f-PCA were observed with the incidence of 5.6% which is in accordance with the previous literature [ 18 - 20 ].

    On the embryological background of CW, the cerebral arteries begin approximately at 5 weeks of gestation. At this stage, many intracranial arteries develop, branch and anastomoses and certain arteries regress among themselves to maintain and to alter towards the adult type of arrangement [ 21 , 22 ]. The anterior part of CW originates from ICA which divides into cranial and caudal arteries while the posterior part from bilateral longitudinal neural arteries. The caudal part forms the PCoA, the carotid-vertebrobasilar communicating artery. Initially PCA represents a branch of primitive ICA which then arises from basilar artery when vertebrobasilar system develops as the occipital lobes enlarge and its functional demand increases [ 23 - 25 ]. Around 22 weeks PCoA or P1 segment of PCA may enlarges to meet the supply of posterior cerebral circulation and there found to be a continuous alteration of blood flow between carotid and vertebrobasilar system until the growth of brain is fulfilled. According to Padget [ 26 ], embryogenesis of CW takes place in two stages, first development of numerous arterial plexuses and regression of certain arterial segments in-utero or postnatal, transforming itself into adult type [ 27 ]. The posterior part of CW being more anomalous and variant, the posterior part of brain benefit from more blood supply when compared to the anterior part [ 28 , 29 ].

    In a study of functional PCoA in posterior circulation ischemia, revealed that the blood flow volume in PCoA can compensate for the decreased flow of BA circulation to a certain degree and play a protective role in posterior cerebral ischemia [ 30 ]. According to literature, a complete absence of PCA was never been reported, but its origin from BA or ICA only varies [ 31 ].

    In the present study bilateral hypoplasia of PCoA was found more than unilateral cases which can be taken into consideration as one of the risk factors for the development of stroke. The percentage of stroke was found to be 60% in case of bilateral absence of PCoA, 40% in unilateral absence of PCoA and 20% in patients having both PCoA [ 16 ]. In union with A1 segment of ACA and VA hypoplasia either unilateral or bilateral PCoA can result in headaches, hypertension, vertigo, pulsatile tinnitus and posterior circulation stroke [ 32 , 33 ].

    The limitation of the study is that, the difficulty to sort out extremely smaller branches which take part in the collateral circulation to rule out the exact diagnosis such as the CIS. The study also included a limited number of patients which in a larger population can provide even much higher rate of variations and a diverse way of diagnostic approach towards PCoA and its associated anomalies.

    Under any major arterial occlusion, the collateral circulation plays a key role and takes over the supply of that deficient vessel thereby reducing the risk of stroke or any occlusive diseases of brain. In such conditions, collateral circulation will be more effective when there is a complete CW with the presence of anterior and PCoA. If any one of these vessels is absent or dysfunction, then collateral circulation will be impaired. In ICA occlusion circulation from the contralateral ICA may be achieved by the presence of patent anterior communicating artery [ 34 ]. Collateral flow is enabled via the PCoA from the vertebra basilar system thereby aiding perfusion.

    Hypoplastic PCoA should be the highly prioritized and monitored arteries in high risk group&rsquos patients with brain tumours, trauma injuries and cardiovascular complications.

    The PCoA, being the main conduit between internal carotid and vertebrobasilar arterial systems, any type of variation of its own and its related structures such as anterior communicating artery PCA or VA as observed in the present study can be a significant record for clinicians and neurosurgeons intended to neurological procedures.


    Watch the video: Internal Carotid Artery - Anatomy Circle of Willis (November 2022).