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Immunohistochemical study of aquaporins in an African grey parrot (Psittacus erithacus) with hydrocephalus.

Abstract: A 5-month-old African grey parrot (Psittacus erithacus) was examined after 3 weeks of weakness, ataxia, mental depression, and seizures. Results of a complete blood cell count and plasma biochemical analysis were unremarkable. Magnetic resonance imaging revealed a severe bilateral hydrocephalus. The bird failed to improve with supportive care, and the owner requested euthanasia. Necropsy findings were severe bilateral hydrocephalus with no evidence of cerebrospinal fluid obstruction. Histologic examination of the brain revealed microspongiosis, edema, gliosis, and neuronal chromatolysis of surrounding periventricular tissue. Aquaporins (AQP) and astrocytes were examined to elucidate the participation of these water channel proteins and glial cells in the pathophysiology and resolution of hydrocephalus. Results showed AQP4 and glial fibrillary acidic protein were overexpressed, especially near the ventricles, but expression of AQP1 was decreased. This is the first report, to our knowledge, of AQP immunolabeling in hydrocephalus in avain species.

Key words: aquaporin, AQP, cerebrospinal fluid, CSF, hydrocephalus, avian, African grey parrot, Psittacus erithacus

Clinical Report

A 5-month-old African grey parrot (Psittacus erithacus) was referred to the Hospital Clinic Veterinari at the Universitat Autonoma de Barcelona for neurologic deficits. The bird had a 3-week history of weakness, mental depression, ataxia, and seizures. Physical examination was unremarkable, except for poor body condition (evaluation score 2/5). Neurologic examination revealed depressed mental status, incoordination, impaired proprioceptive reactions, and visual deficits; the animal also showed difficulty grasping food items. Neurologic examination was consistent with an intracranial disease affecting both cerebral hemispheres. The main differential diagnoses were congenital defects, inflammatory/infectious disease, and metabolic encephalopathies. Complete blood cell count and plasma biochemical results were within reference intervals, ruling out metabolic disease associated with hypocalcemia or hypoglycemia and kidney or liver disease. Results of Chlamydophila polymerase chain reaction and paramyxovirus-3 serologic tests were negative. A brain magnetic resonance imaging with a 0.2-T permanent magnet unit (Vet-MR, Esaote, Italy), performed with the bird under general anesthesia, showed severe dilatation of both lateral ventricles, mainly in the caudal portion of the cerebral hemispheres. The rostral opening of the mesencephalic aqueduct was slightly dilated, but no enlargement of the lateral extensions of the aqueduct or the fourth ventricle was observed (Fig 1). The presumptive diagnosis was congenital hydrocephalus. Because of failure to improve after 2 weeks of conservative management, the owner requested euthanasia.

After a complete necropsy, tissue samples were fixed in 10% buffered formalin and paraffin embedded, and 3-pm sections were used for hematoxylin and eosin and periodic acid-Schiff (PAS) staining.

Further edema-associated protein studies were done with immunohistochemical techniques of aquaporin 4 (AQP4, rabbit, 1:200, Chemicon, Temecula, CA, USA), aquaporin 1 (AQP1, rabbit, 1 : 300, Chemicon) and glial fibrillary acidic protein (GFAP, rabbit, 1:1000, Dako, Glostrup, Denmark). The immunoreaction was amplified with the avidin-biotin complex and visualized with 3,3'-diaminobenzidine. A normal brain of an African grey parrot was used as a control.

The brain showed bilateral enlargement of cerebral hemispheres, generalized ventriculomegaly, and enlargement of the mesencephalic aqueduct. The caudal external surface was thinner than normal. No other macroscopic changes were observed.

Microscopic examination showed bilateral dilatation of the lateral ventricles and mesencephalic aqueduct associated with marked periventricular white mater microspongiosis. Eosinophilic droplets were also observed mainly in the neuropil next to the perivascular spaces. Both occipital cortices showed markedly diminished thickness, diffuse microspongiosis of the neuropil, neuronal chromatolysis, and neuronal death. Gliosis and more reactive astrocytes than usual were seen, some accumulating eosinophilic droplets. Although no point of obstruction was found, congenital noncommunicating hydrocephalus was diagnosed. Intracytoplasmatic material and eosinophilic droplets stained positive for PAS (Fig 2), corresponding to a high proportion of carbohydrate macromolecules contained in those eosinophilic droplets.

In the control brain, immunohistochemical studies showed aquaporins in several cell populations. Aquaporin 4 was present in high amounts in astrocytes and ependymocytes and in low amounts in the choroidal plexus epithelia and leptomeningeal cells. In contrast, AQP1 was present in high amounts in choroidal plexus cells, leptomeningeal cells, and perivascular and cerebellar astrocytes and in low amounts in ependymocytes.

The distribution and intensity of AQP4, AQP1, and GFAP labeling in the hydrocephalic brain of the African grey parrot are summarized in Table 1. Astrocytic positivity for AQP4 was higher than in the control brain, especially in grey matter (Fig 3). Positivity was most intense in the cerebral cortex, the striatum, the hippocampus, and cerebellar grey matter. Fower intensity was observed especially below the glia limitans (inner and outer) and in the neostriatum, and perineuronally in mesencephalic, cerebellar, and medulla oblongata nuclei.

The AQP1 study revealed a marked decrease in the intensity of staining compared with the control brain (Fig 3). Positivity was most pronounced in the most vascularized tissues, such as the grey matter. Positivity was intense in choroid plexus cells, inner and outer glia limitans, and leptomeninges and was moderate in ependymocytes and the perivascular endfeet.

The GFAP immunoreactivity in the hydrocephalic brain was stronger than in the control brain, both in number of cells and intensity (Fig 3). Reactivity in the brainstem was strong in fibrillary astrocytes and scattered in white and grey matter, whereas in the cerebellum, it was intense in Bergmann glia and the perineuronal in Purkinje cells.

The striatum area is the largest structure in the psittacine brain, and it is located directly beside the lateral ventricles. In the hydrocephalic brain, we found that AQP4 and GFAP positivity was higher in the striatum, both in intensity and in number of cells marked than was seen in the control brain. We observed a general correlation in the distribution and intensity of these 2 markers, whereas AQP1 showed less immunolabeling in the hydrocephalic brain than in the control brain.

Discussion

Neurologic signs related to hepatic encephalopathy, renal problems, vitamin deficiencies, and infectious diseases are relatively common problems in pet birds, but hydrocephalus has been infrequently reported in avian species. (1)

Hydrocephalus is caused by an imbalance between the formation and absorption of cerebrospinal fluid (CSF), resulting in fluid accumulation in the brain. This buildup leads to enlargement of the ventricles and progressive damage to brain cells. The disorder can be congenital or acquired. The most common causes of hydrocephalus are developmental disorders. Most acquired cases in small animals result from blockage of CSF outflow secondary to neoplastic or inflammatory conditions. Occasionally, CSF overproduction may occur as the result of choroid plexus tumors. (2)

Most cases of hydrocephalus are described in domestic poultry and are associated with the chemotherapeutic agent cyclophosphamide and ingestion of alkaloids from the plant Veratrum californicum. Congenital defects linked to an autosomal recessive gene in turkeys have also been reported. (3-5) The few cases described in other bird species are all congenital or of unknown cause: an Andean condor (Vultur gryphus), (6) 2 African grey parrots, (7,8) a Goffin's cockatoo (Cacatua goffini), (9) and a yellow-headed Amazon parrot (Amazona ochrocephala oratrix). (10)

We classified the present case as congenital hydrocephalus based on the bird's age, history, the course of the clinical signs, and histopathologic observations, even though no obstructions or choroid plexus alterations were detected. The nonaltered size of the fourth ventricle in our case suggests an obstructive, noncommunicating hydrocephalus that may have been caused by stenosis along the cerebral aqueduct not observed in the sections studied.

Hydrocephalus is associated with major changes in water content and probably extracellular flow. (11) Regulation of brain water movements is critical to maintain normal neuronal function. The increased intracranial pressure in hydrocephalic conditions drives flow from the ventricles to the parenchyma, leading to interstitial edema, especially in subventricular white matter. (12) Cerebral edema in the present case was marked, especially in the perivascular spaces, with an increased endothelial-neuropil space. The PAS-positive droplets in the perivascular spaces corresponded to glycoproteins and were probably caused by the edema formation and to active transport in astrocytes from extracellular to vascular spaces.

Aquaporins are a family of integral membrane proteins that act as water channels, and some of them are involved in CSF movement within the brain. They have been divided into 3 functional groups: aquaporins (permeable to water), aquaglyceroporins (permeable to water, glycerol, and urea), and neutral solute channels (permeable to water, glycerol, urea, purines, pyrimidines, and carboxylates). (13) Two of the aquaporins that have been identified in the brain are AQP1 and AQP4. Aquaporin 1 is mainly expressed in the apical pole of the choroid plexus epithelial cells. Aquaporin 4 is the dominant water channel in the brain, and it is amply distributed, found in astrocyte foot processes, and in contact with blood vessels and the CSF-brain interface (pial and ependymal surfaces). (13) Whereas AQP4 has a widespread astrocytic pattern, AQP1 has a more restricted localization. (14) Studies in humans and other animals, such as nonhuman primates, mice, and rats suggest that AQP1 and AQP4 are involved in the development or maintenance of hydrocephalus, but this has not been studied in avian species. In this study, we mapped the expression of these 2 proteins in an African grey parrot hydrocephalic brain and compared findings with those in a normal African grey parrot brain.

Aquaporin 4 is expressed in supporting cells, such as astrocytes and ependyma, especially in the interface between major fluid compartments and brain parenchyma, but it is not expressed in neurons. In abnormal situations, especially in those associated with brain edema where the upregulation of AQP4 in astrocytes is an important mechanism, there is a redistribution of AQP4 accompanied by a loss of its polarized expression pattern. (15) In this African grey parrot, AQP4 was overexpressed in the whole brain, especially in the striatum, cerebral cortex, hippocampus, cerebellum, and perivascular spaces.

Aquaporin 4 has an important role in resolving edema after brain injury. (16) At the earlier time points (vasogenic edema), AQP4 has a positive effect in preventing edema formation by inhibiting water channels, but later in the course of the disease (cytotoxic edema), it has a key role in water clearance from the brain into blood vessels. Hydrocephalus induces upregulation of AQP4 expression in perivascular astrocytes and capillary endothelia. (11) Excess water is mainly eliminated through the external glia-limiting membranes into the CSF by an AQP4-dependent route. Aquaporin4 knockout mice develop marked hydrocephalus, probably because of reduced water clearance through the ependyma and brain-blood barrier. (15) A recent study (15) has also documented that AQP4 is upregulated in periventricular white matter of hydrocephalic rats. However, how AQP4 collaborates in the clearance of water and fluids from parenchyma to blood vessels is not well understood. In another rat model of obstructive hydrocephalus, overexpression of AQP4 was not observed, neither by immunohistochemical nor by Western blot, suggesting major intracranial pressure or edema is needed to upregulate this protein. (17) In agreement with previous studies, the severe hydrocephalus observed in our case was associated with marked AQP4 overexpression.

Aquaporin 1 mediates water transport in the formation of CSF. It is mainly expressed in the apical pole membranes of the ventricular choroid plexus cells. Astrocytes do not express AQP1 in normal conditions, but in a recent report (13) in a nonhuman primate (Macaca fascicularis), a subtype of astrocytes mainly located in the white matter and the glia limitans did express this protein in the processes and perivascular endfeet. We did not observe any overexpression of AQP1 in our study. These results are in agreement with studies done in humans and in other animal species that showed no evidence of AQP1 upregulation in hydrocephalus, suggesting that overproduction of CSF is not the cause of the hydrocephalus. (18)

In this case, we observed an increased and intense immunolabeling for GFAP in the hydrocephalic brain, indicating astrocytic activation. This astrocytic activation, together with AQP4 upregulation, suggests a crucial role for this cell type and its water channels in improving edema clearance from the brain parenchyma. Despite the AQP4 activation, however, the edema was not resolved in this parrot, and brain damage was irreversible. We conclude that AQPs, especially AQP4, are useful markers for the study of brain edema in avian species.

References

(1.) Jones MP, Orosz SE. Overview of avian neurology and neurological diseases. Semin Avian Exotic Pet Med. 1996;5(3): 150 164.

(2.) Thomas WB. Hydrocephalus in dogs and cats. Vet Clin North Am Small Anim Pract. 2010;40(1): 143-159.

(3.) Bryden MM, Perry C, Keeler RF. Effects of alkaloids of Veratrum californicum on chick embryos. Teratology. 1973;8(1): 19-25.

(4.) Kar AK, Singh S, Sanyal AK. Cyclophosphamide induced hydrocephalus in chick embryos. Indian J Med Res. 1974;62(6):905-908.

(5.) Nestor KE. Hereditary chondrodystrophy in the turkey. Poult Sci. 1978;57(3):577-580.

(6.) Lora-Michiels M, Isaza R, Hoskinson J, et al. Clinical challenge. J Zoo Wildl Med. 2001;32(1): 143 145.

(7.) Wack R, Lindstrom J, Graham D. Internal hydrocephalus in an African grey parrot (Psittacus erithacus timneh). J Assoc Avian Vet. 1989;3(2):94 96.

(8.) Fleming GJ, Lester NV, Stevenson R, Silver XS. High field strength (4.7T) magnetic resonance imaging of hydrocephalus in an African grey parrot (Psittacus erithacus). Vet Radiol Ultrasound. 2003;44(5):542 545.

(9.) Johnston HA, Lindstrom JG, Oglesbee M. Communicating hydrocephalus in a mature Goffin's cockatoo (Cacatua goffini). J Avian Med Surg. 2006;20(3): 180-184.

(10.) Keller KA, Guzman DS, Muthuswamy A, et al. Hydrocephalus in a yellow-headed Amazon parrot (Amazona ochrocephala oralrix). J Avian Med Surg. 2011 ;25(3):216-224.

(11.) Mao X, Enno TL, Del Bigio MR. Aquaporin 4 changes in rat brain with severe hydrocephalus. Eur J Neurosci. 2006;23(11):2929-2936.

(12.) Williams MA, Razumovsky AY. Cerebrospinal fluid circulation, cerebral edema, and intracranial pressure. Curr Opin Neurol. 1993;6(6):847-853.

(13.) Arcienega II, Brunet JF, Bloch J, Badaut J. Cell locations for AQP1, AQP4 and 9 in the non-human primate brain. Neuroscience. 2010; 167(4): 1103 1114.

(14.) Venero JL, Vizuete ML, Machado A, Cano J. Aquaporins in the central nervous system. Prog Neurobiol. 2001;63(3):321-336.

(15.) Loreto C, Reggio E. Aquaporin and vascular diseases. Curr Newopharmacol. 2010;8(2): 105-111.

(16.) Badaut J, Ashwal S, Obenaus A. Aquaporins in cerebrovascular disease: a target for treatment of brain edema. Cerebrovasc Dis. 2011 ;31 (6):521-531.

(17.) Aghayev K, Bal E, Rahimli T, et al. Aquaporin-4 expression is not elevated in mild hydrocephalus. Ada Neurochir (Wien). 2012; 154(4):753-759.

(18.) Albertini R, Bianchi R. Aquaporins and glia. Curr Neuropharmacol. 2010;8(2):84-91.

Ester Blasco, DVM, MSc *, Jaime Martorell, DVM, PhD, Dipl ECZM (Small Mammal) *, Cristian De la Fuente, DVM, PhD, Dipl ECVN, and Marti Pumarola, DVM, PhD, Dipl ECVP

From the Department of Animal Medicine and Surgery, Faculty of Veterinary Science, Universitat Autonoma de Barcelona, Edifici V, Bellaterra 08193, Spain.

* These authors contributed equally to this work.

Table 1. Distribution and intensity of AQP4, AQP1, and GFAP labeling
in the brains of 2 African grey parrots. A control (CTL) brain is
compared with the hydrocephalic (HCP) brain.

Brain area                                   AQP4        AQP1

                                           CTL    HCP    CTL
Striatum
  Hyperstriatum accessorium                 +     +++     ++
  Hyperstriatum intercalatum supremum       +     +++     -
  Neostriatum                              +/-     ++    +/-
  Lamina frontalis superior                +/-     ++     -
  Lamina hyperstriatica                    +/-     ++     -
  Lamina medullaris dorsalis                +     +++     +
  Subventricular zone                       -      -      -
  Neostriatum intermedium                   -      +      -
  Paleostriatum augmentatum                 +      ++     -
  Archistriatum                             +     +++     +
  Fasciculus prosencephalicus lateralis     -      -      -
  Field L                                   -      +      +

Cerebrum
  Hippocampus                               +      ++     -
  Area parahippocampalis                    +     +++     +
  Corticoidea dorsolateralis               +/-     ++     -

Brain stem
  Stratum opticum                           -      -      -
  Tractus septomesencephalicus              -      -      -
  Nucleus taeniae                           +      ++     +

Tectum opticum
  Stratum griseum (SGFS, SGC, SGP)          +     +++     -
  Stratum album centrale                    -      +      -
  Cerebellum                                +      ++     -

Brain area                                 AQP1      GFAP

                                           HCP    CTL    HCP
Striatum
  Hyperstriatum accessorium                 +      +      ++
  Hyperstriatum intercalatum supremum       -      +      ++
  Neostriatum                               ++     +     +++
  Lamina frontalis superior                 +      +      ++
  Lamina hyperstriatica                     +      +      ++
  Lamina medullaris dorsalis               +++     +      ++
  Subventricular zone                       -      -      -
  Neostriatum intermedium                   +     +/-     +
  Paleostriatum augmentatum                +/-     +      ++
  Archistriatum                             ++     +      ++
  Fasciculus prosencephalicus lateralis     +      +      ++
  Field L                                   ++     +      ++

Cerebrum
  Hippocampus                               +      -      -
  Area parahippocampalis                    ++     +      ++
  Corticoidea dorsolateralis               +/-    +/-     +

Brain stem
  Stratum opticum                           +     +/-     +
  Tractus septomesencephalicus              +            +/-
  Nucleus taeniae                           ++     +      ++

Tectum opticum
  Stratum griseum (SGFS, SGC, SGP)          +      +      ++
  Stratum album centrale                    -     +/-     +
  Cerebellum                                +      +      ++

Abbreviations: AQP, aquaporin; GFAP, glial fibrillary acidic protein;
+, moderate; ++, strong positive; +++, very strong positive; -,
negative; SGFS, stratum griseum et fibrosum superficiale; SGC, stratum
griseum centrale; SGP, stratum griseum periventriculare.
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Article Details
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Title Annotation:Clinical Reports
Author:Blasco, Ester; Martorell, Jaime; De la Fuente, Cristian; Pumarola, Marti
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:4EUSP
Date:Dec 1, 2014
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