The Vasculature of Nurse Cells Infected with Non-Encapsulated Trichinella Sp

THE VASCULATURE OF NURSE CELLS INFECTED WITH NON-ENCAPSULATED TRICHINELLA SP THE VASCULATURE OF NURSE CELLS INFECTED WITH NON-ENCAPSULATED TRICHINELLA SPECIES Pathamet Khositharattanakool, Nimit Morakote, Pichart Uparanukraw Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Abstract. The vasculature surrounding the nurse cells of encapsulated Trichi- nella spiralis has been described previously. It has been postulated the function of these vessels is to support the growth of the parasite. We describe here for the first time the vasculature surrounding the nurse cells of non-encapsulated T. pseudospiralis and T. papuae. Similar to the vasculature of uninfected muscle cells, the vessels surrounding non-encapsulated Trichinella nurse cells are dense and branched longitudinally along the long axis of the muscle cells; they also appear to be similar in diameter. The netting pattern of enlarged vessels found around T. spiralis (encapsulated) nurse cells is not present in non-encapsulated Trichinella infections. The vessels surrounding non-encapsulated Trichinella nurse cells seem to exist prior to parasite invasion of the muscle cell. Keywords: Trichinella, nurse cell, vasculature INTRODUCTION and replacement by rough endoplasmic reticulum (Despommier, 1976; Chang Trichinella is an intracellular parasitic et al, 1988; Jasmer, 1993), enlargement and nematode that infects skeletal muscle. division of nurse cell nuclei (Chang et al, Infection occurs by ingesting meat con- 1988; Despommier, 1993; Jasmer, 1993), taminated with the infective stage of mitochondrial damage by vacuolation Trichinella larvae. After the larvae develop (Despommier, 1975), and formation of col- into adults in the host intestine, mating oc- lagen capsule surrounding the nurse cell curs and gravid females release newborn (Polvere et al, 1997). The collagen capsule larvae into the blood stream resulting in of the T. spiralis nurse cell is lemon-shaped the spread of the larvae throughout the and composed of collagen types IV and VI body. The larva infects muscle cells and (Polvere et al, 1997; Nareaho, 2006). The transforms them into nurse cells which T. spiralis larva is coiled and firmly en- support the growth of the parasite. closed in the collagen capsule of the There are many changes in the for- nurse cell (Al Karmi and Faubert, 1981). mation of the nurse cell infected with T. The presence or absence of a collagen spiralis, including loss of myofilaments capsule is used to distinguish the species of Trichinella as either encapsulated or Correspondence: Pichart Uparanukraw, De- non-encapsulated species, respectively partment of Parasitology, Faculty of Medicine, (Zarlenga et al, 2006). Chiang Mai University, Chiang Mai 50200, Thailand. Non-encapsulated species of T. pseu- Tel: + 66 (0) 53 945342; Fax: + 66 (0) 53 217144 dospiralis and T. papuae are able to form E-mail: [email protected] nurse cells. The obvious differences Vol 44 No. 4 July 2013 561 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH between the nurse cell of T. spiralis and ents and disposal of waste products (De- the non-encapsulated Trichinella species spommier, 1998). Until now, there have are the latter do not form a collagen cap- been no studies demonstrating the pres- sule and the cytoplasmic changes occur ence of this vascular network surrounding throughout the length of the infected non-encapsulated species of Trichinella muscle cell which is more elongated than nurse cells. In this study, we attempted that of T. spiralis (Al Karmi and Faubert, to delineate the vasculature of muscle 1981; Chang et al, 1988; Pozio et al, 1999; cells infected with non-encapsulated Wu et al, 2001; Boonmars et al, 2005). T. pseudospiralis and T. papuae. A feature of the cytoplasm of the T. pseudospiralis nurse cell is eosinophilic MATERIALS AND METHODS staining with H&E stain, in contrast to the basophilic staining of the T. spiralis Trichinella infected mice nurse cell (Matsuo et al, 2000; Wu et al, Imprinting control region (ICR) mice 2001; Boonmars et al, 2004). More acid were orally infected with 250 L1 stage T. phosphatase activity is observed in the spiralis or T. pseudospiralis larvae. The T. nurse cells of T. spiralis than in those of T. spiralis used in this study was isolated pseudospiralis and is probably associated from a human in Mae Hong Son, Thai- with muscle cell damage (Boonmars et al, land, in 1986 (Pozio and Khamboonruang, 2004). Loss of myofilaments, increased 1989). The laboratory strain of T. pseudo- number of endoplasmic reticulum and spiralis used in this study was generously mitochondrial damage are found in the provided by Professor Yuzo Takahashi, nurse cells of both T. pseudospiralis and Gifu University, Japan. These parasites T. papuae (Pozio et al, 1999). The nuclei were maintained in ICR mice. The mice of non-encapsulated Trichinella nurse were raised at Animal Laboratory House, cells are smaller than those of T. spiralis- Faculty of Medicine, Chiang Mai Univer- infected nurse cells. Non-encapsulated sity, Thailand. Swiss mice orally infected larva appears both coiled and uncoiled with 40 L1 T. papuae larvae were kindly and moves freely in the nurse cell (Al provided by Professor Pewpan Malee- Karmi and Faubert, 1981). wong, Khon Kaen University, Thailand. This study was approved by the Animal One of the remarkable features of T. Research Ethics Committee, Faculty of spiralis nurse cells is the vascular network Medicine, Chiang Mai University. surrounding the collagen capsule. The vascular network is a net of vessels sur- Muscle perfusion with ink rounding the nurse cell which can be seen Ink perfusion for the observation by injection of China ink (Ogielski, 1949) of blood vessels surrounding Trichinella and by the intravascular precipitation of nurse cells was conducted using the modi- lead chromate (Humes and Akers, 1952). fied method of Baruch and Despommier Blood vessel studies using an anatomical (1991). The mice were infected with Trichi- plastic cast in muscles infected with T. spi- nella at least 3 months prior to the study. ralis show details of the vascular network The Trichinella-infected mice were eutha- structure similar to a randomly woven nized by intraperitoneal injection with 0.3 basket or rete (Baruch and Despommier, ml of sodium pentothal at a concentration 1991). These vessels have been postulated of 65 mg/ml. The chest cavity was opened to function in the transportation of nutri- and a hole was made in the right atrium. 562 Vol 44 No. 4 July 2013 THE VASCULATURE OF NURSE CELLS INFECTED WITH NON-ENCAPSULATED TRICHINELLA SP Heparinized saline (500 U of heparin/ml) range from 4 to 6 µm in width. was then immediately instilled into the T. spiralis-infected nurse cells are left ventricle and flushing was continued found to have blood vessels surrounding until the solution flowing from the right the collagen capsule in a netting pattern atrium was clear when the 50% Rotring or rete (Figs 1B, 2A and 2B). Some vessels drawing ink (black) in saline was injected are seen in the collagen layer (Fig 2A). until the ears, feet and tail turned gray. The diameters of the vessels surrounding The muscles infused with ink were surgi- the nurse cells are generally larger than cally removed and the vasculature of the those in the uninfected muscles (Figs 1B, Trichinella-infected muscles was studied 2A and 2B). The sizes of the vessels in the using a muscle compression technique, rete range from 4 to 12 µm. under an Olympus AX70TF differential Examination of T. pseudospiralis and interference contrast (DIC) microscope T. papuae infected muscles reveals that or an Olympus BX51 DIC microscope. the vessels supplying the nurse cells are The blood vessels adjacent to the infected dense and run along the length of the muscle surface in the same focal plane muscle (Fig 1C and 1D). The sizes of the were then measured. vessels are 4 to 6 µm in diameter and their Histological study branching patterns appear to be similar to Trichinella-infected gastrocnemius those of adjacent uninfected muscle cells. muscles perfused with drawing ink were Unlike T. spiralis-infected muscles, no net- fixed with 10% buffered formalin and de- ting patterns of vessels are seen around hydrated using increasing concentrations T. pseudospiralis and T. papuae infected of alcohol: 80% ethanol for 30 minutes muscle cells. Paraffin sections confirm twice, 95% ethanol for 20 minutes three that the vessels surrounding muscle cells times and 100% ethanol for 20 minutes infected with T. pseudospiralis seem to three times. The muscles were cleared be not different from those surrounding with xylene for 20 minutes three times. uninfected muscle cells (Fig 2C and 2D). The specimens were then placed in melted paraffin at 60ºC for 2 hours. The paraffin- DISCUSSION embedded muscles were sectioned at 5 µm thick and placed on silanized slides. It has been known for some time an- The sections were stained with H&E stain giogenesis leads to the vascular network according to conventional method and ex- around nurse cells infected with T. spiralis; amined under the microscope (Olympus occurs during nurse cell formation (Ogiel- AX70TF DIC). ski, 1949; Humes and Akers, 1952; Baruch and Despommier, 1991). In this study, RESULTS we describe the vasculature of nurse cells infected with non-encapsulated T. pseudo- The normal vasculature of skeletal spiralis and T. papuae. There are differences muscle sections is shown in Fig 1A. The between the vasculature of encapsulated vessels of uninfected muscle are dense and non-encapsulated Trichinella-infected and longitudinally branched along the muscles. Nurse cells in T. spiralis infection long axis of the muscle fiber. The vessels are surrounded by a network of blood supplying the muscle cells are branches of vessels with an average diameter larger the larger vessels. The sizes of the vessels than those of uninfected muscles. Humes Vol 44 No. 4 July 2013 563 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH Fig 1–Photomicrograph of the vasculature perfused with drawing ink in an uninfected muscle cell (A), T.

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