I also say it is good to fall, battles are lost in the same spirit in which they are won. And to all generals that lost engagements, and all overcome heroes! And the numberless unknown heroes equal to the greatest heroes known! It is for the wicked just the same as the righteous, I make appoint- ments with all,.
This is the press of a bashful hand, this the float and odor of hair,. Well I have, for the Fourth-month showers have, and the mica on the side of a rock has. Does the daylight astonish? Whimpering and truckling fold with powders for invalids, con- formity goes to the fourth-remov'd,. Having pried through the strata, analyzed to a hair, counsel'd with doctors and calculated close,. In all people I see myself, none more and not one a barley-corn less,.
I know this orbit of mine cannot be swept by a carpenter's compass,. I know I shall not pass like a child's carlacue cut with a burnt stick at night. I reckon I behave no prouder than the level I plant my house by, after all.
'68 (Sixty-Eight): Scars #1 (of 4) - Comics by comiXology
One world is aware and by far the largest to me, and that is my- self,. And whether I come to my own to-day or in ten thousand or ten million years,. I can cheerfully take it now, or with equal cheerfulness I can wait. The pleasures of heaven are with me and the pains of hell are with me,. The first I graft and increase upon myself, the latter I translate into a new tongue. It is a trifle, they will more than arrive there every one, and still pass on. Press close bare-bosom'd night—press close magnetic nourishing night!
Earth of the vitreous pour of the full moon just tinged with blue! Earth of the limpid gray of clouds brighter and clearer for my sake! Prodigal, you have given me love—therefore I to you give love! We must have a turn together, I undress, hurry me out of sight of the land,. Sea of the brine of life and of unshovell'd yet always-ready graves,.
Partaker of influx and efflux I, extoller of hate and conciliation,. Shall I make my list of things in the house and skip the house that supports them? I am not the poet of goodness only, I do not decline to be the poet of wickedness also. Evil propels me and reform of evil propels me, I stand indifferent,. Did you guess the celestial laws are yet to be work'd over and rectified? What behaved well in the past or behaves well to-day is not such a wonder,.
The wonder is always and always how there can be a mean man or an infidel. Here or henceforward it is all the same to me, I accept Time abso- lutely. This is the lexicographer, this the chemist, this made a grammar of the old cartouches,. This is the geologist, this works with the scalpel, and this is a mathematician. And more the reminders they of life untold, and of freedom and extrication,. And make short account of neuters and geldings, and favor men and women fully equipt,. And beat the gong of revolt, and stop with fugitives and them that plot and conspire.
No sentimentalist, no stander above men and women or apart from them,. Through me the afflatus surging and surging, through me the cur- rent and index. By God! I will accept nothing which all cannot have their coun- terpart of on the same terms. And of the threads that connect the stars, and of wombs and of the father-stuff,. I keep as delicate around the bowels as around the head and heart,.
Seeing, hearing, feeling, are miracles, and each part and tag of me is a miracle. Divine am I inside and out, and I make holy whatever I touch or am touch'd from,. If I worship one thing more than another it shall be the spread of my own body, or any part of it,. Root of wash'd sweet-flag! Winds whose soft-tickling genitals rub against me it shall be you! Broad muscular fields, branches of live oak, loving lounger in my winding paths, it shall be you!
Hands I have taken, face I have kiss'd, mortal I have ever touch'd, it shall be you. I cannot tell how my ankles bend, nor whence the cause of my faintest wish,. Nor the cause of the friendship I emit, nor the cause of the friend- ship I take again. A morning-glory at my window satisfies me more than the meta- physics of books.
Hefts of the moving world at innocent gambols silently rising freshly exuding,. The earth by the sky staid with, the daily close of their junction,. We found our own O my soul in the calm and cool of the day- break. With the twirl of my tongue I encompass worlds and volumes of worlds. Come now I will not be tantalized, you conceive too much of articulation,. My knowledge my live parts, it keeping tally with the meaning of all things,.
Happiness, which whoever hears me let him or her set out in search of this day. My final merit I refuse you, I refuse putting from me what I really am,. To accrue what I hear into this song, to let sounds contribute toward it. I hear bravuras of birds, bustle of growing wheat, gossip of flames, clack of sticks cooking my meals,. Sounds of the city and sounds out of the city, sounds of the day and night,.
Talkative young ones to those that like them, the loud laugh of work-people at their meals,. The angry base of disjointed friendship, the faint tones of the sick,. The judge with hands tight to the desk, his pallid lips pronoun- cing a death-sentence,. The heave'e'yo of stevedores unlading ships by the wharves, the refrain of the anchor-lifters,. The ring of alarm-bells, the cry of fire, the whirr of swift-streak- ing engines and hose-carts with premonitory tinkles and color'd lights,. The steam-whistle, the solid roll of the train of approaching cars,.
The slow march play'd at the head of the association marching two and two,. They go to guard some corpse, the flag-tops are draped with black muslin. I hear the violoncello, 'tis the young man's heart's complaint,.
It wrenches such ardors from me I did not know I possess'd them,. It sails me, I dab with bare feet, they are lick'd by the indolent waves,. Steep'd amid honey'd morphine, my windpipe throttled in fakes of death,. If nothing lay more develop'd the quahaug in its callous shell were enough. To touch my person to some one else's is about as much as I can stand. My flesh and blood playing out lightning to strike what is hardly different from myself,.
Deluding my confusion with the calm of the sunlight and pasture- fields,. They bribed to swap off with touch and go and graze at the edges of me,. I talk wildly, I have lost my wits, I and nobody else am the greatest traitor,. I went myself first to the headland, my own hands carried me there. You villain touch! Blind loving wrestling touch, sheath'd hooded sharp-tooth'd touch!
Reactive astrocytes function as phagocytes after brain ischemia via ABCA1-mediated pathway
Sprouts take and accumulate, stand by the curb prolific and vital,. And a summit and flower there is the feeling they have for each other,. And they are to branch boundlessly out of that lesson until it becomes omnific,. I believe a leaf of grass is no less than the journey-work of the stars,.
And the pismire is equally perfect, and a grain of sand, and the egg of the wren,. I find I incorporate gneiss, coal, long-threaded moss, fruits, grains, esculent roots,. In vain the plutonic rocks send their old heat against my approach,. In vain the ocean settling in hollows and the great monsters lying low,. I follow quickly, I ascend to the nest in the fissure of the cliff.
I think I could turn and live with animals, they are so placid and self-contain'd,. Not one is dissatisfied, not one is demented with the mania of owning things,. Not one kneels to another, nor to his kind that lived thousands of years ago,. They bring me tokens of myself, they evince them plainly in their possession. Picking out here one that I love, and now go with him on brotherly terms. A gigantic beauty of a stallion, fresh and responsive to my caresses,. Eyes full of sparkling wickedness, ears finely cut, flexibly moving. His well-built limbs tremble with pleasure as we race around and return.
And again as I walk'd the beach under the paling stars of the morning. By the city's quadrangular houses—in log huts, camping with lumbermen,. Along the ruts of the turnpike, along the dry gulch and rivulet bed,. Weeding my onion-patch or hoeing rows of carrots and parsnips, crossing savannas, trailing in forests,. Scorch'd ankle-deep by the hot sand, hauling my boat down the shallow river,. Where the panther walks to and fro on a limb overhead, where the buck turns furiously at the hunter,.
Where the rattlesnake suns his flabby length on a rock, where the otter is feeding on fish,. Where the black bear is searching for roots or honey, where the beaver pats the mud with his paddle-shaped tail;. Over the growing sugar, over the yellow-flower'd cotton plant, over the rice in its low moist field,. Over the sharp-peak'd farm house, with its scallop'd scum and slender shoots from the gutters,.
Over the western persimmon, over the long-leav'd corn, over the delicate blue-flower flax,. Over the white and brown buckwheat, a hummer and buzzer there with the rest,. Over the dusky green of the rye as it ripples and shades in the breeze;. Scaling mountains, pulling myself cautiously up, holding on by low scragged limbs,.
Walking the path worn in the grass and beat through the leaves of the brush,. Where the bat flies in the Seventh-month eve, where the great gold- bug drops through the dark,. Where the brook puts out of the roots of the old tree and flows to the meadow,. Where cattle stand and shake away flies with the tremulous shud- dering of their hides,.
Where the cheese-cloth hangs in the kitchen, where andirons straddle the hearth-slab, where cobwebs fall in festoons from the rafters;. Where trip-hammers crash, where the press is whirling its cylinders,.
Post-Liver Transplant Clinic
Wherever the human heart beats with terrible throes under its ribs,. Where the pear-shaped balloon is floating aloft, floating in it my- self and looking composedly down,. Where the life-car is drawn on the slip-noose, where the heat hatches pale-green eggs in the dented sand,. Where the fin of the shark cuts like a black chip out of the water,. Where shells grow to her slimy deck, where the dead are corrupt- ing below;.
Where the dense-starr'd flag is borne at the head of the regiments,. Under Niagara, the cataract falling like a veil over my countenance,. Upon the race-course, or enjoying picnics or jigs or a good game of base-ball,. At he-festivals, with blackguard gibes, ironical license, bull-dances, drinking, laughter,. At the cider-mill tasting the sweets of the brown mash, sucking the juice through a straw,.
At musters, beach-parties, friendly bees, huskings, house-raisings;. Where the mocking-bird sounds his delicious gurgles, cackles, screams, weeps,. Where the hay-rick stands in the barn-yard, where the dry-stalks are scatter'd, where the brood-cow waits in the hovel,. Where the bull advances to do his masculine work, where the stud to the mare, where the cock is treading the hen,. Where the heifers browse, where geese nip their food with short jerks,. Where sun-down shadows lengthen over the limitless and lonesome prairie,. Where herds of buffalo make a crawling spread of the square miles far and near,.
Where the humming-bird shimmers, where the neck of the long- lived swan is curving and winding,. Where the laughing-gull scoots by the shore, where she laughs her near-human laugh,. Where bee-hives range on a gray bench in the garden half hid by the high weeds,.
Where band-neck'd partridges roost in a ring on the ground with their heads out,. Where the yellow-crown'd heron comes to the edge of the marsh at night and feeds upon small crabs,. Where the katy-did works her chromatic reed on the walnut-tree over the well,. Through patches of citrons and cucumbers with silver-wired leaves,. Through the gymnasium, through the curtain'd saloon, through the office or public hall;. Pleas'd with the native and pleas'd with the foreign, pleas'd with the new and old,. Pleas'd with the quakeress as she puts off her bonnet and talks melodiously,. Pleas'd with the earnest words of the sweating Methodist preach- er, impress'd seriously at the camp-meeting;.
- How To Seed Grass;
- Alien UFO.
- The Texas Rangers.
Looking in at the shop-windows of Broadway the whole forenoon, flatting the flesh of my nose on the thick plate glass,. Wandering the same afternoon with my face turn'd up to the clouds, or down a lane or along the beach,. My right and left arms round the sides of two friends, and I in the middle;. Coming home with the silent and dark-cheek'd bush-boy, behind me he rides at the drape of the day,.
Far from the settlements studying the print of animals' feet, or the moccasin print,. By the cot in the hospital reaching lemonade to a feverish patient,. Nigh the coffin'd corpse when all is still, examining with a candle;. Solitary at midnight in my back yard, my thoughts gone from me a long while,. Walking the old hills of Judaea with the beautiful gentle God by my side,.
Speeding amid the seven satellites and the broad ring, and the diameter of eighty thousand miles,. Carrying the crescent child that carries its own full mother in its belly,. My messengers continually cruise away or bring their returns to me. I go hunting polar furs and the seal, leaping chasms with a pike- pointed staff, clinging to topples of brittle and blue. Through the clear atmosphere I stretch around on the wonderful beauty,. The enormous masses of ice pass me and I pass them, the scenery is plain in all directions,. The white-topt mountains show in the distance, I fling out my fancies toward them,.
We are approaching some great battle-field in which we are soon to be engaged,. We pass the colossal outposts of the encampment, we pass with still feet and caution,. The blocks and fallen architecture more than all the living cities of the globe. My voice is the wife's voice, the screech by the rail of the stairs,. How the skipper saw the crowded and rudderless wreck of the steam-ship, and Death chasing it up and down the storm,.
Pre-Liver Transplant Clinic
How he knuckled tight and gave not back an inch, and was faith ful of days and faithful of nights,. And chalk'd in large letters on a board, Be of good cheer, we will not desert you;. How he follow'd with them and tack'd with them three days and would not give it up,. How the lank loose-gown'd women look'd when boated from the side of their prepared graves,. How the silent old-faced infants and the lifted sick, and the sharp- lipp'd unshaved men;.
All this I swallow, it tastes good, I like it well, it becomes mine,. The mother of old, condemn'd for a witch, burnt with dry wood, her children gazing on,. The hounded slave that flags in the race, leans by the fence, blow- ing, cover'd with sweat,. The twinges that sting like needles his legs and neck, the mur- derous buckshot and the bullets,. Hell and despair are upon me, crack and again crack the marks- men,. I clutch the rails of the fence, my gore dribs, thinn'd with the ooze of my skin,. Taunt my dizzy ears and beat me violently over the head with whip-stocks.
I do not ask the wounded person how he feels, I myself become the wounded person,. Although the cancer first starts within the liver, as it grows it can spread to other organs, a process called metastasis. HCC most frequently spreads to the lungs or to bones. The risk of spread outside of the liver increases with the size of the cancer. Liver transplantation definitively cures a patient of HCC, provided that the tumor has not spread beyond the liver. Because there are far more people in need of liver transplants than there are available organs, specific guidelines, called the Milan Criteria, have been established to define which patients with HCC are eligible for transplantation.
These criteria define limits of tumor number and size that ensure a very low likelihood of cancer spread outside of the liver. There are many people with cirrhosis and decompensated liver disease but not all are appropriate candidates for liver transplantation. A patient must be able to survive the operation and the potential post-operative complications, reliably take the medications that prevent rejection and opportunistic infections, comply with frequent clinic visits and laboratory tests, and not engage in activity that would injure the liver, such as drinking alcohol.
The conditions listed below are generally considered to be absolute contra-indications to liver transplantation. Allocation policy determines how any available organs will be distributed among the many candidates on the waiting list. Over the past five years, the number of patients awaiting a liver transplant has been largely unchanged.
Our current allocation policy is guided by the principles outlined by the Final Rule, issued by the Department of Health and Human Services in March The Rule stipulates that allocation policy should give primary consideration to the urgency of a recipient's need for transplantation. This has come to be known as the concept of "sickest first. Much research has gone into trying to understand how to accurately determine how sick a person is from his or her liver disease. A scoring system, called MELD Model for End-stage Liver Disease , has been identified as highly predictive of the risk of death posed by chronic liver disease.
The MELD score is determined by the results of three objective and readily available laboratory tests:. The patient with the highest MELD score the sickest patient is therefore at the top of the list. Lists are, however, organized by blood type. When a donor liver becomes available, the blood type of the donor is determined and the person at the top of the list for that blood group is offered the organ. If that person is too sick or does not accept the liver for whatever reason, the liver is then offered to the next person on the list and so forth until a suitable recipient is found.
Another complicating factor in liver allocation policy is geography. The United States is divided into 11 regions and each region, in turn, is divided into multiple donor service areas DSAs. The DSAs are the smallest or the "local" unit of organ allocation. Most frequently, organs that are procured from donors in a specific DSA are allocated to candidate recipients within the same DSA. This policy arose as an approach to minimize times for organ transportation and preservation. The Final Rule, however, emphasized the importance of disease severity and discounted the impact of geography in organ allocation policy.
Most livers used for transplantation are obtained from patients that are brain dead. The trauma has stopped all brain function although other organs including the liver may continue to function normally. There are strict definitions as to what constitutes brain death based on the complete absence of any type of brain function. Because patients that meet criteria for brain death are legally dead, they are appropriate organ and tissue donors. In other countries, such as France, consent for organ donation is presumed and allowed, unless the family objects.
Typically, transplant centers whose patients will be receiving organs from a particular donor will dispatch a team of surgeons to procure the relevant organ. The organ procurement procedure takes place in an operating room in the donor's hospital. Organs are removed and preserved in a fashion to optimize their condition during the storage and transportation time period. Each procured organ is then transported to the hospitals where the designated recipient awaits. Sometimes a patient suffers a devastating brain injury and carries a dismal neurological prognosis but fails to meet the strict criteria defining brain death in that there is still detectible brain function.
In these circumstances, the patient's family may decide to withdraw life-sustaining medical support with the intention of allowing the patient to die. In this scenario, death is not defined by brain death but rather cardiac death. Organ donation can occur after cardiac death but, again, only if the family gives consent.
Only AFTER the family's decision to withdraw support may the patient be considered for organ donation after death. Under these circumstances, support is withdrawn, as desired by the family and managed by the patient's physician, and the patient is allowed to expire. He or she then declares the patient's death. An urgent operation is then performed to preserve and remove organs for transplantation. This mode of cardiac death, in contrast to brain death, results in increased injury to the organs during two time periods.
The first period is that between withdrawal of life support and death. As the donor's breathing and circulation deteriorates, the organs may no longer be receiving sufficient oxygen. The second time period constitutes the minutes immediately after death and until the organs are flushed with preservation solution and cooled. As a result, livers procured from cardiac death donors are associated with an increased risk of primary non function or poor early organ function, hepatic artery thrombosis, and biliary complications see Complications section. Although each person has only one liver and would die without it, it is possible to donate a portion of the liver for transplantation into another individual.
The segmental anatomy see figure below allows surgeons to create grafts of varying size, depending upon the recipient's requirement for liver tissue. The partial livers in both the donor and the recipient will grow to provide normal liver function for both individuals. Historically, this procedure was developed to enable transplantation of children as it was difficult to find suitable livers from deceased donors for this group.
An adult-to-adult living donor liver transplant is highly complex and technically challenging procedure that carries a significant risk for both the donor and the recipient. A liver transplant involves the removal of and preparation of the donor liver, removal of the diseased liver, and implantation of the new organ. The liver has several key connections that must be re-established for the new organ to receive blood flow and to drain bile from the liver.
The structures that must be reconnected are the inferior vena cava, the portal vein, the hepatic artery, and the bile duct. The exact method of connecting these structures varies depending on specific donor and anatomy or recipient anatomic issues and, in some cases, the recipient disease. For someone undergoing liver transplantation, the sequence of events in the operating room is as follows:. As with any surgical procedure, complications related to the operation may occur, in addition to the many possible complications that may happen to any patient who is hospitalized.
Some of the problems specific to liver transplantation that may be encountered include:. If the function of the liver does not improve sufficiently or quickly enough, the patient may urgently require a second transplant to survive. The human body has developed a very sophisticated series of defenses against bacteria, viruses, and tumors. The machinery of the immune system has evolved over millions of years to identify and attack anything that is foreign or not "self.
A number of drugs are given to transplant recipients to dampen the responses of their immune system in an attempt to keep the organ safe and free of immunologic attack. If the immune system is not sufficiently weakened, then rejection - the process by which the immune system identifies, attacks, and injures the transplanted organ - ensues. Commonly used drugs to prevent rejection by suppressing the immune system are listed below. They work through different mechanisms to weaken the immune system's responses to stimuli and are associated with different side effects.
As a result, these medications are frequently used in various combinations which increase the overall immunosuppressive effect while minimizing side effects. Rejection is a term that is applied to organ dysfunction caused by the recipient's immune system reaction to the transplanted organ. Injury to the liver is typically mediated by immune cells, T cells or T lymphocytes.
Rejection typically causes no symptoms; patients do not feel any differently or notice anything. The first sign is usually abnormally elevated liver laboratory test results. When rejection is suspected, a liver biopsy is performed. Liver biopsies are easily done as a bedside procedure using a special needle that is introduced through the skin. The tissue is then analyzed and inspected under the microscope to determine the pattern of liver injury and also to look for the presence of immune cells. Once the diagnosis is made, treatment is fairly straightforward and generally very effective.
The first line of treatment is high dose corticosteroids see Immunosuppression section. The patient's maintenance immunosuppression regimen is also escalated to prevent subsequent rejection. The second line of rejection treatment is strong antibody preparations see Immunosuppression Section. This is believed to be because the liver has the unique ability to regenerate when injured thereby restoring full liver function.
Liver biopsy shows loss of bile ducts and obliteration of small arteries. Chronic rejection, historically, has been difficult to reverse, often necessitating repeat liver transplantation. Today, with our large selection of immunosuppressive drugs, chronic rejection is more often reversible. Some of the processes that led to the failure of the patient's own liver can damage the new liver and eventually destroy it. Perhaps the best example is hepatitis B infection. The vast majority of these patients suffered from very aggressive reinfection of the new liver by hepatitis B virus.
During the 's, however, several drugs and strategies to prevent re-infection and damage of the new liver were developed and instituted widely by transplant centers. These approaches have been highly successful such that recurrent disease is no longer a problem. Hepatitis B, once considered a contra-indication to transplantation, is now associated with excellent outcomes, superior to many of the other indications for liver transplantation.
Currently, our primary problem with recurrent disease is focused on hepatitis C. Any patient that enters transplantation with hepatitis C virus circulating in their blood will have ongoing hepatitis C after transplantation. However, those who have completely cleared their virus and do not have measurable hepatitis C in the blood will not have hepatitis C after transplantation. Unlike hepatitis B where recurrent disease leading to liver failure occurs very rapidly, recurrent hepatitis C typically causes a more gradual attrition of liver function.
Most have more gradually progressive disease such that as many as half will have cirrhosis at approximately 10 years after transplant. Interferon preparations in combination with ribavirin, widely used in pre-transplant hepatitis C patients, can also be prescribed after transplantation. Chances for permanent cure are somewhat lower than treatment before transplantation. Moreover, the treatment is associated with a significant complement of side effects.
Recurrent disease is responsible for the fact that hepatitis C liver transplant recipients have worse medium and long-term post-transplant outcomes compared to liver transplant recipients without hepatitis C Figure 8. Several other diseases may also recur after transplantation, but typically the disease is mild and only slowly progressive.
Together these findings clearly showed that cultured astrocytes were phagocytic. These characteristic phagocytosis features in astrocytes were similar to those observed in phagocytes. Furthermore, for easier quantitative analysis, fluorescent beads were used as a substrate for phagocytosis 11 , Our results showed that the fluorescent beads could be successfully ingested as a pseudo substrate and the above-mentioned inhibitors inhibited uptake Supplementary Fig. These inhibitors also decreased synaptosome uptake by astrocytes, indicating that cultured astrocytes have the ability to phagocytize apoptotic neurons, surrogate beads, and synaptic debris in our experimental model Supplementary Fig.
To determine the involvement of ABCA1 in astrocytic phagocytosis, we initially used a pharmacological approach. Pretreatment of cultured astrocytes with Glyburide 34 or PSC 45 , which are functional inhibitors of ABCA1, significantly reduced uptake of fluorescent beads in a concentration-dependent manner Fig. ABCA1 mediates astrocytic phagocytosis in vitro. Arrowheads indicate ABCA1 accumulation and points of attachment between captured beads and astrocytes. Forty-eight images per z-stack images 0.
We also demonstrated the involvement of other molecules in the ABCA1 pathway. It is well known that the liver X receptor LXR and retinoid X receptor form a heterodimer that binds to the proximal promoter of the ABCA1 gene, resulting in increased gene transcription Taken together, ABCA1 upregulation itself can enhance engulfment in cultured astrocytes. We analyzed the phagocytic ability of astrocytes based on Galectin-3 and LAMP2 immunoreactivities and found that the intensity of both markers was significantly lower than in littermate controls Supplementary Fig.
Interestingly, astrocyte processes frequently touched or were very close to large debris Figs. The densities of engulfed small debris in astrocytes and microglia were comparable in control mice Fig. Taken together, these data demonstrated that phagocytic astrocytes also actively participate in the remodeling of damaged tissues as well as microglia and that phagocytic signaling through ABCA1 is one main molecular mechanism by which reactive astrocytes engulf debris.
ABCA1 mediates astrocytic phagocytosis in vivo. Ischemic penumbra of control contains extracellular debris a , green , arrows , and astrocyte processes a , b , left , blue with glycogen granules a , asterisks include cellular debris a , b , red , arrowheads. By contrast, ischemic penumbra of cKO contains numerous extracellular debris a , green , arrows , but the astrocyte processes a , b , yellow rarely contains debris.
Mann—Whitney U- test. We demonstrated that adult astrocytes can function as phagocytes in a restricted spatiotemporal pattern after transient brain ischemia, and ABCA1 and its related pathway molecules play indispensable roles in this mechanism. Our results revealed the following points. Conversely, phagocytic microglia were evident in the ischemic core region during the early phase after injury. Overall, we propose a new role for reactive astrocytes as phagocytes in pathophysiological condition. Astrocytes could work cooperatively with microglia and contribute to the engulfment of dead cells or debris.
However, because there are differences in spatiotemporal phagocytosis patterns between astrocytes and microglia, astrocytes play a distinct role in clearance of damaged tissue from microglia. Results suggest that the phagocytic astrocytes likely contribute to remodeling and recovery of the brain microenvironment within the ischemic penumbra region Supplementary Fig. EM analyses showed many phagosomes in astrocytes in the ipsilateral striatum after ischemic injury, but detected very few or no phagosomes in astrocytes in healthy or contralateral striatum, respectively Figs.
However, previous studies reported that adult astrocytes in the healthy optic nerve head myelination transition zone actively phagocytose axonal debris 17 , 18 , and astrocytes in the cortex are involved in synapse phagocytosis This suggests that astrocytic phagocytosis in the healthy adult brain could occur in several brain regions, but its frequency seems to be partly brain-region-dependent and is low in the striatum.
In contrast to array tomography analysis 20 , our EM observations allowed the high-resolution analysis of a small area. This might explain why we failed to detect a low frequency astrocytic phagocytosis in the healthy adult striatum. During development, astrocytes show intense phagocytic activity of synapses in the lateral geniculate nucleus Although these astrocytes might engulf synapses in the cortex of adult brains, but the efficacy is much lower than during developmental stages During brain development, network structures and functions dynamically change as well as after acute brain injury, and the brain becomes more plastic than normal adult brain under these situations.
Accumulating evidence indicates both astrocytes and microglia play roles in remodeling during these dynamical plastic changes 7 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , Together, these findings suggest that astrocytes can phagocytize adjacent materials, including synapses, although this occurs primarily during development or under pathophysiological conditions in the adult brain. Further investigation might indicate whether adult astrocytes also participate in network remodeling via engulfment under physiological situations such as learning and memory.
Recent findings revealed that neuroinflammation and ischemia induced different types of reactive astrocytes termed A1 harmful and A2 protective , respectively 21 , In this literature, the focus was limited to the functions of A1 reactive astrocytes, which have deficient synapse engulfment in the developmental lateral geniculate nucleus. Although this literature have not examine the functions of A2 reactive astrocytes yet, the heterogeneous activations of astrocytes should be taken into account when discussing other brain diseases.
To date, two redundant pathways have been reported to have a role in phagocytosis 31 In fact, in cultures, suppression and overexpression of these molecules resulted in inhibition and facilitation of astrocytic phagocytosis, respectively Fig. Additionally, the knockdown of both Megf10 and Gulp1 significantly decreased astrocytic phagocytosis Supplementary Fig. ABCA1 is a member of the ABC transporter family and known to transport lipid species, such as cholesterol and phospholipid, across the membrane bilayer Although the exact function of ABCA1 in engulfment has not been clearly revealed yet, there are various roles proposed, e.
It is not known how ABCA1 contributes to the astrocytic phagocytosis observed in the present study, but according to the notion observed above, it is possible that it is involved in the recognition or engulfment of debris. On the other hand, we did not clearly show the involvement of second pathway. According to Chung et al. Thus, astrocytic phagocytosis might partly share similar pathways during developmental stages and under pathological conditions. It remains poorly understood why phagocytic cells have multiple phagocytosis-related molecules, including phagocytic receptors and intracellular signaling molecules.
Recent finding showed that astrocyte-like glial cells eliminate neuronal subcompartments via context-dependent use of distinct engulfment pathways during larval metamorphosis in Drosophila This suggests that Drosophila astrocytes use these molecules differently depending on their targets. If this is also the case for mammalian astrocytes, the ABCA1-dependent astrocytic phagocytosis observed in this study might be specific for special targets.
Unlike astrocytes, microglial engulfment was evident during the early phase within the ischemic core region Figs. This rapid clearance of dying cells by professional phagocytes could prevent diffusion of detrimental contents owing to subsequent loss of permeability of the cell membranes.
Results from previous studies suggested that the kinetics of phagocytosis are different between professional and non-professional phagocytes, i. Even under pathological conditions, astrocytes were not as mobile as microglia 26 , 64 suggesting astrocytes might not be involved in the acute clearance of damaged tissue in the ischemic core region. In addition, 3D-EM observations revealed no large phagocytic inclusions such as whole degenerating neurons in astrocyte cytoplasm although their processes were attached to large debris.
This suggests that there might be a limit to the size of debris that can be engulfed by astrocytes. The volume and number of astrocyte processes are much higher compared with microglia; therefore, the amount of debris engulfed by astrocytes would be substantial in the penumbra. The onset of astrocytic phagocytosis began at 3 days and persisted for 2 weeks after MCAO within the ischemic penumbra region Figs. Interestingly, LAMP2 upregulation in astrocytes was broader in areas distal from the ischemic core; however, CD68 upregulation in microglia was limited to the ischemic core and proximal region Fig.
The spatiotemporal pattern of astrocytic phagocytosis suggests a relationship to neuronal remodeling in the ischemic penumbra region 7 , There are substantial axonal, dendritic and synaptic losses and eliminations within the penumbra region within the first week after stroke, and this is followed by an increased number of synapses and axonal connections 6. The present study demonstrated that synaptic debris became incorporated into reactive astrocytes in the penumbra region, and that cultured astrocytes actively phagocytized synaptosomes Supplementary Figs.
These findings support our notion that reactive astrocytes also engage in synapse elimination in the penumbra region. However, we are not proposing that astrocytes are the only cell type that can eliminate synapses after ischemia. Microglia are known to eliminate synapses during developmental stages and pathological conditions 50 , 51 , 56 , Further studies are needed to provide a better understanding of the physiological consequences of astrocytic phagocytosis, and to better elucidate the difference between phagocytosis in astrocytes and microglia. In conclusion, we demonstrated that astrocytes become phagocytic after brain ischemia, and ABCA1 and its pathway molecules play a pivotal role in this process.
The spatiotemporal pattern of astrocytic phagocytosis after stroke, i. To date, studies have focused on the mechanisms involved in microglial phagocytic events, and astrocytes have received very limited attention. Results from this study provide novel information about reactive astrocytes and their role in phagocytic events following ischemic injury in the brain and can hopefully be applied to other brain diseases.
Mice were housed no more than five per cage. Changes in cerebral surface blood flow were monitored by using a laser speckle blood flow imaging system Omegazone, Omegawave, Tokyo, Japan , which obtains high-resolution, two-dimensional imaging and has a linear relationship with absolute CBF as described previously Recordings were performed through the skull under 1. For each recording, the skull surface was wiped with saline-soaked gauze. The CBF was measured in identically sized regions of interest 0.
The mice were deeply anesthetized with pentobarbital and transcardially perfused with PBS containing 0. Sections were permeabilized with 0. Double labeling with in situ hybridization, horseradish peroxidase HRP -conjugated anti-rabbit or anti-mouse IgG 1: ; Medical and Biological Laboratories for secondary antibodies.
To avoid overquenching IHC signals, reaction time in 0. The slices were mounted with DPX mountant Sigma. All reactions were gently shaken on a shaker. Then, sections were washed twice with maleic acid buffer 0. For double staining, IHC was performed following in situ hybridization. After fixation with 2. Following observation under a light microscope and identification of the ischemic penumbra, which is typically located in the striatum near the corpus callosum and cortex, tissue pieces including the penumbra regions were collected for tissue preparation for SBF-SEM imaging.
En bloc heavy metal staining was performed as reported previously with some modifications Blocks from each group were trimmed and mounted on aluminum rivets with conductive glue CW, Circuitworks. The surfaces of the trimmed samples were treated with gold sputtering to increase conductivity, and imaged under various imaging conditions in Merlin or Sigma Carl Zeiss equipped with 3View Gatan.
For detection of ABCA1, the preparations were not boiled to avoid aggregation. The medium was replaced every 3 days until the cells were confluent. At that time, the culture consisted mostly of astrocytes. Half of the culture medium was replaced every 3—4 days. Synaptosomes were purified from adult mice brains by sucrose gradient multiple centrifugation. The pellet consisting of crude synaptosomes was resuspended in PBS.
Primary astrocytes were incubated with fluorescently labeled targets, i. After the indicated time, the cells were extensively washed three times with cold PBS, incubated with 0. Detached neuronal debris, fluorescent beads and synaptosomes were excluded for sampling using forward and side-scattered plots based on their homogeneity and small size compared with astrocytes. For each point, —10, events were collected and the data was analyzed using Cell Quest. The phagocytic index was calculated by geometric mean of fluorescence intensity multiplied by the percentage of fluorescent-positive cells.
Prior to transfection, the astrocytes were fed fresh medium without antibiotics. Allstar negative control siRNA was obtained from Qiagen. All siRNA sequences were non-disclosure. The cultures were washed three times with PBS and then fluorescent images were obtained using a confocal laser scanning system FV; Olympus. Information about z-stack images was described in the figure legends. Images without descriptions are single plane images. The ischemic core was identified as the inside leading edge according to GFAP or 3PGDH immunoreactivity, whereas the penumbra region was defined as the region surrounding the core.
Microglia were imaged based on Iba1 immunostaining. For each section, two or three fields were imaged in the defined space. Immunofluorescence intensity and cell counts were analyzed while the investigator was blind to the experimental conditions. For Fig.
Processes of microglia or astrocytes were analyzed based on Iba1 or Gal-3, GFAP double-positive immunoreactivity, respectively. Colocalization analyses were performed with the Colocalization Threshold Image J plugin. The number of imaging fields, animals used n is indicated in the figure legends, and there were always three or more separate animals for each experiment in all assays. The original version of this Article contained an error in the spelling of the author Nobuhiko Ohno, which was incorrectly given as Noubuhiko Ohno. Murphy, T.
Plasticity during stroke recovery: from synapse to behaviour. Sofroniew, M. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. Anderson, M.
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Astrocyte scar formation aids central nervous system axon regeneration. Nature , — Hobohm, C. Decomposition and long-lasting downregulation of extracellular matrix in perineuronal nets induced by focal cerebral ischemia in rats. Bauer, S. The neuropoietic cytokine family in development, plasticity, disease and injury.