Neural repair in an insect: cell recruitment and deployment following selective glial disruption

Summary In Periplaneta americana, SEM of abdominal nervous connectives revealed a rapid accumulation of haemocytes on the surface of the neural lamella within 24 h of selective disruption of the underlying neuroglia by ethidium bromide. After 4 days the neural lamella was effectively clear of adhering haemocytes, but showed characteristic blisters, which, it is postulated, represented the points of entry of the cells from the haemocoel into the underlying tissues. A notable subsequent feature was a substantial increase in the number of cells within repairing connectives. Initially, there was a marked asymmetry in their distribution, with significantly higher numbers of cells anterior to, and within, the lesion area. It seems likely that this polarity resulted from differential cell division within the connectives. The initial asymmetry disappeared after seven days. However, increased perineurial cell numbers were maintained in the lesion area after one month and were still apparent two months after selective glial disruption. There was no equivalent increase in cell numbers in the lesion zone of cultured cords or, in vivo, after injection of the DNA-scission drug, bleomycin, treatments which preclude haemocyte involvement. It is suggested that in the absence of haemocytes and with suppression of proliferation by endogenous cells, repair is achieved by redeployment or growth of adjacent, undamaged glia.     

Glial repair in an insect

The repair of cockroach central nervous connectives, following selective glial disruption, involves an initial invasion of the lesion by a novel cell class. The available evidence, including that obtained using monoclonal antibodies, shows that these cells arise from circulating haemocytes. These invasive exogenous cells are restricted to the lesion zone. They are not only involved in initial repair of the peripheral glial elements, but may also be responsible for initiating recruitment and division of endogenous reactive cells. There is a clear anterior polarity in this recruitment, with significantly higher numbers of cells appearing anterior to, and then within, the lesion area. Characteristically, recognizable exogenous cells decline in number after 3 days, although there is no overall reduction in cell numbers within the lesion at this stage, nor has significant cell division begun. This suggests that the haemocyte-derived cells transform into, or are replaced by, functional perineurial glia, between 3 and 5 days, coincident with the restoration of the blood-brain barrier and the onset of endogenous cell division. Glial repair in the insect CNS can thus be divided into three phases which show striking similarities to the repair sequence in vertebrate brain. These include: an initial invasion of the lesion by exogenous cells, subsequent glial proliferation and then longer term fluxes in cell numbers and distribution.     

The effects of an anti-mitotic drug,bleomycin, on glial repair in an insect central nervous system

Summary The DNA-binding drug, bleomycin, has a profound effect on neural repair following selective glial disruption by ethidium bromide. The contribution of the granule-containing cells (which normally appear in the early stages of repair) is greatly reduced, the restoration of the blood-brain barrier is delayed and the ultrastructural organization of the reorganising perineurium is dramatically changed. The aberrant perineurial structure and function observed in the presence of bleomycin are postulated to result from the effects of the drug on haemocytes which, together with endogenous reactive cells, contribute to the normal process of glial repair.     

Adult insect glial culture: Activation, substrate effects and proliferation

Glial cells from an adult insect, Periplaneta americana, have been grown in neurone-free cultures. No growth occurred from freshly-excised fragments of abdominal nervous connectives. Vigorous growth was obtained, however, from explants of connectives induced to proliferate by prior exposure to a toxin, ethidium bromide, applied selectively to glial cells in vivo. Glial growth in vitro is dependent upon the initiation of early stages of repair in vivo: this supports the idea that haemocytes which invade the lesion zone immediately after damage are involved in directing proliferation of perineurial and sub-perineurial glia. In contrast, both glial and neuronal cells grew in vitro from explanted abdominal ganglia without prior glial lesioning, indicating that different factors may determine cellular regeneration in this domain. The morphology of the proliferating cells was influenced by the substrate; extensive glial migration was restricted to areas of close contact between cell and substrate surface.     

Glial repair in the cultured central nervous system of an insect

Summary Insect glial cells are capable of division and repair in organ culture after selective damage with the toxin ethidium bromide. The repair is slower and less organised than seen in vivo after similar treatment and is still incomplete after one month. Granule-containing cells, which play an important role in the early stages of repair in vivo, are never seen in cultured connectives. This observation adds further support to the hypothesis that these cells are derived from haemocytes and that their presence is necessary for rapid and orderly repair. The uptake of ³H-thymidine into perineurial glial cells in vitro, both in control and ethidiumtreated connectives, shows that there is a considerable proliferation of cells in this region. Some uptake of thymidine is also seen in subperineurial glia but division alone cannot account for the large increase in the number of glial nuclei found at the early stages of repair in this region. Further, glial cells with diverse morphologies suggest that subpopulations are present. We conclude that cell migration from undamaged areas, as well as cell proliferation, is necessary for CNS repair in vitro.     

Neural repair in an insect central nervous system: cell kinetics and proliferation after selective glial disruption

Summary Autoradiographs of tritiated thymidine uptake and subsequent light- and electron-microscopical examination revealed an onset of perineurial glial cell proliferation 3 days after injury to the CNS. The number of cells labelled increased rapidly until 7 days post-lesioning. At 2 weeks, the labelled cells equalled the number of nuclei present in the perineurium. No label was seen in the subperineurial cells, possibly because of the inability of the label to penetrate into a region where localised division is taking place.Prior to the onset of thymidine uptake, the damaged nerve cord was invaded by an exogenous reactive cell. The number of these cells increased rapidly in the first 48 h, then decreased as a negative exponential, very few remaining after 7 days. We suggest that this cell type must either return to the haemocoel or transform into a functional glial cell class.The repair of the insect central nervous system can be divided into three phases which show striking similarities to vertebrate repair sequences. These include: initial invasion of the lesion by exogenous cells, subsequent proliferation of glial cells, the longer term flux of cell numbers, their distribution and the time scale of events. This suggests that the insect CNS might provide a system for examining common cellular mechanisms and events.     

Blood cells contribute to glial repair in an insect

Using antibodies specific for haemocytes, we have shown that these blood cells penetrate the abdominal nervous connectives of the cockroach following selective disruption of the glia using the DNA-intercalating drug, ethidium bromide, as a glial toxin. Within 4 days post-lesion, the labelled cells formed a mosaic beneath the neural lamella and penetrated deeply among the disrupted subperineurial glia. These observations confirm that exogenous cells are involved in glial repair and support a previous hypothesis that they play critical roles in both structural repair and the recruitment of endogenous reactive cells.     

Endogenous lesions, S-phase-independent spontaneous mutations, and evolutionary strategies for base excision repair

We calculate from published levels of endogenous base lesions that our cells constantly generate and excise during base excision repair (BER) about one million lesions per day. Repair glycosylases may also non-specifically excise an additional number of undamaged bases. The resulting abasic sites are repaired daily by BER. The fidelity of polymerase-beta is 2.4 × 10⁻⁵ and one must postulate additional fidelity mechanisms in the BER complex to explain the low mutation rate of resting cells. Any strategy which constitutively increases glycosylase activity to prevent endogenous lesions from entering S-phase and becoming mutations will also serve to increase the number of mutations per day caused by non-specific excision of normal undamaged bases. The best break-even strategy for reducing endogenous lesion-induced mutations is clearly not one of avid repair. Lower organisms from bacteriophage to fungi have adopted strategies to generate 0.0033 consequential mutations per cell division, no more and no less. Strategies such as down regulating glycosylase activity outside of S-phase to reduce time-dependent mutation frequency while leaving lesion replication-induced mutation frequency unchanged are discussed.     

Human TE671 cells express functional motilin receptors

In our search for a cell line expressing endogenous human motilin receptor, we have discovered that theTE671 cell line, a neuron-derived medulloblastoma human line, expresses functional motilin receptors. The cDNA of the receptor was isolated from the cells and its sequence was confirmed to be identical to the previously reported cDNA sequence isolated from human thyroid. The function of the receptor protein was evaluated both for its ability to inhibit the binding of ¹²⁵I-motilin to a crude membrane preparation of TE671 cells and for activation of the phospholipase C signal transduction pathway by calcium mobilization assay. The precise numbers of motilin receptor RNA molecule in TE671 cell and 24 human tissues were quantitatively determined by real-time PCR. TE671 cell line should be a useful tool for the study of motilin receptor-involved signal transduction in humans.     

Brain hormone carrier haemocytes in the moth Monema flavescens

The movement of neurosecretory substances released from the neurosecretory B cell in the pars intercerebralis to the haemolymph was examined with the progress of the termination of diapause in the slug moth pharate pupa, Monema flavescens.The injection of precipitates in the haemolymph of the pharate pupa just before the termination of diapause into diapausing pharate pupae reduced the numbers of days required for them to pupate. In the precipitates, seven types of haemocytes were present. The number of haemocytes, especially the granular cell, increased just before the termination of diapause. AF and CHP positive substances not detected in the haemocytes of diapausing pharate pupae appeared in the granular cells just before the termination of diapause. The period also coincided well with the releasing period of the neurosecretory B cell. Histological examination showed that granular haemocytes gathered around the pars intercerebralis at this period and exchange of neurosecretory substances occurred between granular haemocytes and neurosecretory B cells. Then granular haemocytes migrated to the region of the prothoracic gland. From digestion tests of the neurosecretory substances with rabbit serum and from the implantation tests of the neuroendocrine system, the substances detected in both the neurosecretory B cell and the granular haemocytes seemed to be the same. The dye injection caused a delay in larval-pupal ecdysis emergence. Droplets of black ink are incorporated into the granular haemocytes. This seems to be caused by blocking of the transport of neurosecretory substances released from cytoplasmic processes of the neurosecretory B cell.From these experiments, it is suggested that neurosecretory substances of the prothoracotropic hormone are transported to the prothoracic gland, along with granular haemocytes, after being released directly from the neurosecretory B cell to the haemolymph.     

Intranasal HB-EGF administration favors adult SVZ cell mobilization to demyelinated lesions in mouse corpus callosum

In the adult rodent brain, the subventricular zone (SVZ) represents a special niche for neural stem cells; these cells proliferate and generate neural progenitors. Most of these migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into interneurons. SVZ-derived progenitors can also be recruited spontaneously to damaged brain areas to replace lost cells, including oligodendrocytes in demyelinated lesions. In this study, we searched for factors able to enhance this spontaneous recruitment of endogenous progenitors. Previous studies have suggested that epidermal growth factor (EGF) could stimulate proliferation, migration, and glial differentiation of SVZ progenitors. In the present study we examined EGF influence on endogenous SVZ cell participation to brain repair in the context of demyelinated lesions. We induced a focal demyelinated lesion in the corpus callosum by lysolecithin injection and showed that intranasal heparin-binding epidermal growth factor (HB-EGF) administration induces a significant increase in SVZ cell proliferation together with a stronger SVZ cell mobilization toward the lesions. Besides, HB-EGF causes a shift of SVZ-derived progenitor cell differentiation toward the astrocytic lineage. However, due to the threefold increase in cell recruitment by EGF treatment, the absolute number of SVZ-derived oligodendrocytes in the lesion of treated mice is higher than in controls. These results suggest that enhancing SVZ cell proliferation could be part of future strategies to promote SVZ progenitor cell mobilization toward brain lesions.     

Infection with the protozoan parasite Bonamia ostreae modifies in vitro haemocyte activities of flat oyster Ostrea edulis

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy.Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.     

Classification of haematopoietic cells and haemocytes in Chinese prawn Fenneropenaeus chinensis

It is commonly believed that crustacean haemocytes originate from a specialised haematopoietic tissue (HPT), whereas the differentiation relationship between HPT cells and circulating haemocytes is still not clearly understood. The HPT cells and haemocytes of Fenneropenaeus chinensis were characterised using morphological and histochemical methods. Three types of HPT cells were identified under the transmission electron microscope (TEM). Type 1 cells had high N/C ratios, developed dispersed chromatins and no cytoplasmic granules. Type 2 cells had smaller size, developed condensed chromatins and cytoplasmic granules, which were homogeneous or striated in type 2a cells, and homogeneous in type 2b cells. We deduce that type 1 cells may give rise to type 2 cells in terms of the presence of possible intermediates between type 1 and type 2 cells. The circulating haemocytes were divided into three populations, i.e. hyaline haemocytes (HH), small granular haemocytes (SHG) and large granular haemocytes (LGH), based on Wright-Giemsa staining and TEM observation. Comparing the HPT cells with the circulating haemocytes, type 2a cells of HPT may represent the HH due to similar granule types, cell size and N/C ratios, and type 2b cells may be the young and immature LGH. By Wright-Giemsa and acid alpha-naphthyl acetate esterase staining, the intermediates between the HH and SGH were observed, which indicates that the SGH may be derived from the HH in the circulatory system. Therefore, it is suggested that the F. chinensis haemocytes could be divided into two haemocyte lineages, i.e. the HH-SGH and LGH lineage.     

In experimental ricin intoxication there is a detectable gastric acid secretion in the rat.

According to previous investigations, ricin the toxic protein of castor oil seeds (Ricinus communis, Euphorbiaceae) given in subtoxic dose, evokes a parathyroid lesion with a consecutive parathormone mobilization in the rat. The elevated parathormone level rises the plasma calcium-content resulting (among others) in endogenous gastrin mobilization. This latter increases the gastric acid secretion in significant degree. This, hitherto unknown effect of ricin directs the attention once again to the complexity of its toxic action.     

Correlations between adrenal weight and heart weight in rats with a cardiac overload

A significant positive correlation between heart weight and adrenal weight was found in rats with myocardial hypertrophy induced by experimental hyperthyroidism or ligation of the abdominal aorta. The simultaneous administration of digitoxin partly inhibited myocardial hypertrophy after ligation of the abdominal aorta, but not after experimental hyperthyroidism. Digitoxin also inhibited adrenal hypertrophy after ligature of the abdominal aorta but, again, not after experimental hyperthyroidism. The possible existence of an endogenous cardiotropic hormone participating in the development of cardiac hypertrophy from overloading is discussed.     

Plant polyphenols mobilize nuclear copper in human peripheral lymphocytes leading to oxidatively generated DNA breakage: Implications for an anticancer mechanism

It was earlier proposed that an important anti-cancer mechanism of plant polyphenols may involve mobilization of endogenous copper ions, possibly chromatin-bound copper and the consequent pro-oxidant action. This paper shows that plant polyphenols are able to mobilize nuclear copper in human lymphocytes, leading to degradation of cellular DNA. A cellular system of lymphocytes isolated from human peripheral blood and comet assay was used for this purpose. Incubation of lymphocytes with neocuproine (a cell membrane permeable copper chelator) inhibited DNA degradation in intact lymphocytes. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. This study has further shown that polyphenols are able to degrade DNA in cell nuclei and that such DNA degradation is inhibited by neocuproine as well as bathocuproine (both of which are able to permeate the nuclear pore complex), suggesting that nuclear copper is mobilized in this reaction. Pre-incubation of lymphocyte nuclei with polyphenols indicates that it is capable of traversing the nuclear membrane. This study has also shown that polyphenols generate oxidative stress in lymphocyte nuclei which is inhibited by scavengers of reactive oxygen species (ROS) and neocuproine. These results indicate that the generation of ROS occurs through mobilization of nuclear copper resulting in oxidatively generated DNA breakage.     

Chymotryptic-type protease inhibitors block the increase in Ca2+ and Il-2 production in activated Jurkat T cells

Several chymotryptic-type protease inhibitors were found to inhibit both anti-CD3 mAb- and PHA-induced rise in Ca2+ and IL-2 production in Jurkat T cells. The magnitude of inhibition was a function of the effectors used to stimulate Ca2+ entry and depended on the concentration of the inhibitors. Neither tryptic-type protease inhibitors nor an elastase substrate prevented anti-CD3 mAb- or PHA-induced Ca2+ rise in Jurkat cells. The inhibitory effect of N-alpha-p-tosyl-L-phenylalanine chloromethyl-ketone on anti-CD3 mAb- and PHA-induced rise in Ca2+ resulted from a rapid increase in Ca2+ efflux. The inhibitors which were effective on Ca2+ mobilization also inhibited IL-2 production initiated by an anti-CD3 mAb in the presence of 12-O-tetradecanoylphorbol-13-acetate, and to a lesser extent by PHA or the calcium ionophore A23187. No inhibition of IL-2 production was observed when tryptic-type protease inhibitors or the elastase inhibitor were used. In addition, membrane preparations from Jurkat cells were found to hydrolyze the chymotryptic substrate Suc-Ala-Ala-Phe-paranitroaniline, an effect markedly inhibited by N-alpha-p-tosyl-L-phenylalanine chloromethylketone. Moreover, this inhibitor protected one potential endogenous substrate (Mr 38 kDa) from proteolysis. Taken together, these observations show that chymotryptic-type protease inhibitors block the responses generated by the binding of anti-CD3 mAb to Jurkat cells, and suggest that a chymotryptic-like membrane protease contributes to T cell activation.     

Comparative ultrastructure and blood-brain barrier in diapause and nondiapause larvae of the European corn borer Ostrinia nubilalis (Hübner)

Ultrastructural examination of diapause and nondiapause larval brains of the European corn borer disclosed anatomical differences that may be related to the insect's "blood-barrier". The perineurial type I cells are quite closely appressed in the diapause brain, but thrown into extensive folds with large intercellular spaces in the nondiapause brain. The perineurial type II cells of diapause and nondiapause larvae are basically similar in general ultrastructure, and most likely form the basis for the "blood-brain barrier." Horseradish peroxidase penetration studies indicated that the outer margin of the perineurial type II cells constitute the limits of infiltration into the brain. An enzymatic component of the "blood-brain barrier" is postulated in this insect. The localization of ATPase in the perineurial type II cells indicates that energy-requiring regulatory mechanisms may be localized here. Metabolic studies with isolated brains, coupled with recent evidence from mammalian systems, suggest that glial cells may be of importance in an enzymatic "blood-brain barrier."