Big Ass White Mature
Chrystal Imaizumi
White adipose tissue (WAT) is a central factor in the development of type 2 diabetes, but there is a paucity of translational models to study mature adipocytes. We describe a method for the culture of mature white adipocytes under a permeable membrane. Compared to existing culture methods, MAAC (membrane mature adipocyte aggregate cultures) better maintain adipogenic gene expression, do not dedifferentiate, display reduced hypoxia, and remain functional after long-term culture. Subcutaneous and visceral adipocytes cultured as MAAC retain depot-specific gene expression, and adipocytes from both lean and obese patients can be cultured. Importantly, we show that rosiglitazone treatment or PGC1α overexpression in mature white adipocytes induces a brown fat transcriptional program, providing direct evidence that human adipocytes can transdifferentiate into brown-like adipocytes. Together, these data show that MAAC are a versatile tool for studying phenotypic changes of mature adipocytes and provide an improved translational model for drug development.
Methods: 82 patients who had white mature cataract in one eye, posterior subcapsular, cortical, nuclear, or mixed type cataract in the other eye were enrolled in this prospective study. Postoperative outcomes, intraoperative difficulties related to capsulorhexis, and phacoemulsification were analysed between the two groups of eyes. Postoperative examinations were done at 1 day, 1 week, 1 and 3 months.
Results: Postoperative visual acuity, central corneal thickness, intraocular pressure, and rate of posterior capsule rupture were not significantly different between the two group of eyes (p>0.05). Mean effective phaco time, frequency of postoperative corneal oedema and posterior capsular plaque were found to be significantly higher in the mature cataract group (p
Cyclopentolate 1% and phenylephrine 2.5% eye drops were used for mydriasis four times 1 hour before the surgery. Endocapsular phacoemulsification was performed in all eyes by using Diplomax (Allergan) phacoemulsification unit by one of the three surgeons in an identical manner. Topical or retrobulbar anaesthesia was used. Pieces of cell sponge which are impregnated with oxybuprocaine (proparacaine) HCl 0.5% (Alcaine, Alcon) were placed deep into the superior and inferior fornix for 15 minutes before surgery for topical anaesthesia. Retrobulbar injection was performed using a 23 gauge needle; 3 ml of 2% lidocaine was given intraconally. A three step clear corneal tunnel incision was made with a 3.2 mm disposable metal blade and a side port incision was made with an MVR blade. After injection of sodium chondroitin sulfate-sodium hyaluronate (Viscoat, Alcon) into the anterior chamber of eyes with white cataracts 0.1 ml of trypan blue 0.1% (trypan blue 0.4% in 0.81% sodium chloride solution, cell culture tested, Sigma; diluted with BSS) was injected onto the anterior capsule slowly. Simultaneously the dye was spread mechanically with the anterior chamber cannula to obtain a homogeneous and diffuse staining of the anterior capsule. Additional sodium chondroitin sulfate-sodium hyaluronate was given in order to deepen the anterior chamber and obtain anterior chamber clarity. The dye was used for all white mature cataracts which lacked red fundus reflex. A central initial puncture of the anterior capsule was made with a 26 gauge needle, and the needle was moved to the left approximately 2.0 mm in order to create a capsular flap. Uttrata forceps were used to grasp the capsule and perform a capsulorhexis of 5.0 mm diameter. Gentle hydrodissection was made and the nucleus was rotated with an anterior chamber cannula. If the capsulorhexis tear was directed towards the periphery more viscoelastic was given onto the anterior capsule at that location or Vannas scissors was used to prevent uncontrolled extension. Endocapsular phacoemulsification was performed using a quadrant divide and conquer technique in eyes with white cataracts, chip and flip technique in contralateral eyes. A 15 degree bevel tip was used for all cases. Cortical remnants were removed by an automated irrigating/aspiration hand piece of the phaco unit, anterior and posterior capsular polishing was performed. Corneal incision was enlarged to 4.0 mm with a metal blade, a foldable hydrophilic acrylic intraocular lens with a 12.0 mm overall size and a 5.75 mm optic (Soft Tec I, Lenstec) was implanted intracapsularly and the viscoelastic was removed. The anterior chamber was inflated using a balanced salt solution. The corneal incision was checked for water tightness and left unsutured. Postoperatively all patients used ofloxacin 0.3% and prednisolone acetate 1% eye drops four times a day for 1 month.
In all cases in the mature cataract group the anterior capsule stained adequately for visibility during capsulorhexis. Slight staining of the corneal and side port incision site cleared after irrigation/aspiration of cortical material. No residual coloration of the posterior capsule was noted after removal of lens material. In the mature cataract group capsulorhexis was performed successfully using only Uttrata forceps and without the need of additional viscoelastic in 69 eyes. In 13 cases which developed uncontrolled extension of capsulorhexis more viscoelastic was given onto the capsule or Vannas scissors was used to prevent the tear edge going to periphery. Despite to that, peripheral extension to the equator occurred in two eyes; we returned to the starting point of the capsulorhexis, proceeded with a second tear in the opposite direction and completed the capsulorhexis. Capsulorhexis was successfully completed using only Uttrata forceps and without additional viscoelastic in all eyes in the control group.
General recommendations for visualisation of the anterior capsule in eyes with white mature cataracts include dimming the operation room lights, increasing the magnification of microscope, using oblique illumination, using an endoilluminator,8 capsule dyes, and performing a two stage capsulorhexis.1,2,4,9 In our cases, staining the capsule with trypan blue under viscoelastic material enhanced the visualisation of the anterior capsule during capsulorhexis. Although originally Melles et al10 stained the capsule under an air bubble it was reported that using the dye under a dispersive viscoelastic material was easier and safe.11 The air bubble technique was reported to be time consuming and it was difficult to reform the anterior chamber by a single air bubble.11 Neither viscoelastic exchange nor irrigation was required with the concentration of trypan blue we used. We injected only a small amount of additional viscoelastic material into the anterior chamber to replace the portion of viscoelastic material that escaped during capsule staining manipulation and to obtain anterior chamber clarity. Viscoelastic exchange and irrigation steps prolong surgery and require extra anterior chamber manipulations. Additionally, the enhanced visualisation of the anterior capsule helped the surgeon to identify the capsule edge from the underlying white cortical material and avoid operating trauma to the capsulorhexis edge during phacoemulsification.
The difficulties in performing capsulorhexis in eyes with mature cataracts were absence of red fundus reflex, leakage of fluid immediately as the capsule is punctured, fragility of anterior capsule. Additionally intracapsular pressure remains high even after the initial puncture and the tear tends to extend to the periphery and it becomes difficult to control the capsulorhexis.3 We achieved a one stage, 5 mm continuous capsulorhexis in most of the cases.
Although hydrodissection was not recommended in white mature cataracts,1 we observed that gentle hydrodissection broke the corticocapsular adhesions that could resist free nucleus rotation. Singh et al12 reported corticocapsular adhesions resulted in difficult nucleus rotation in brunescent and black cataracts. Nucleus rotation is crucial for phacoemulsification.
In our cases we did not encounter complications of capsular fibrosis and geometrical decentration. In another study capsular fibrosis was reported to occur in 12% of eyes with white mature cataracts, all of which had a capsulorhexis diameter of less than 5 mm.2 Small capsulorhexis was reported to lead to capsule contraction.13,14
White cataract cases presumed to be of intumescent subtype were included in the study. The other subtypes like white pearly cataracts associated with the solid cortex, morgagnian cataracts, white cataracts with calcified or fibrosed anterior capsule were eliminated from the study. The preoperative clues for the intumescent subtypes were (i) On slit-lamp biomicroscopy these cases had the presence of a shallow AC, presence of fluid vacuoles or sectoral markings in the anterior cortex [Fig. 1a], (ii) On USG A-scan white cataracts showing the presence of multiple internal acoustic reflections were categorized as the intumescent subtype, where the multiple spikes indicated multiple fluid compartments [Fig. 1b]. On the other hand, the cases which showed a few internal acoustic reflections on USG A-scan [Fig. 1c] and no slit-lamp findings indicative of the intumescent subset were excluded from the study. With the recent introduction of newer Swept-source-based optical coherence tomography (SS-OCT) devices such as CASIA 2 (Tomey, Nagoya, Japan) the full thickness scanning of the lens has become possible. The intralenticular fluid pockets can be viewed preoperatively and help in differentiating the intumescent subtypes from the other subsets of white mature cataracts [Fig. 1d]. However, this modality was introduced later on as a part of the assessment, therefore the related findings were not included in the study.
(a) An intumescent cataract with shallow anterior chamber (blue arrow), with translucent sectoral markings in the cortex (yellow arrows) and presence of a translucent fluid-filled area between the anterior lens capsule and the lens fibers (red arrow) (b) USG A-Scan of the eye in figure a, showing multiple intralenticular spikes (c) A pearly white cataract showing few intralenticular spikes on USG A-Scan (d) SS-OCT of the eye described in figure a and b, showing shallow anterior chamber with anteriorly bulging, swollen lens and multiple hypoechoic areas indicating cortical fluid clefts
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