The heterogeneity of the entities grouped as NBIA has been a limiting factor in the evaluation of neuropathological data [20
]. Since the recognition of PKAN as a genetically homogeneous disease, however, it has been possible to retrospectively identify reports of patients likely to have had PKAN based on clinical and radiographic data. This has supported the delineation of neuropathologic findings in PKAN.
NBIA was a postmortem diagnosis prior to the availability of MRI as a diagnostic tool. On gross sectioning, the globus pallidus and reticular zone of the substantia nigra show rust-brown pigmentation mainly composed of iron [19
]. Routine iron stains detect the metal mainly in the microglia and macrophages, but scattered neurons are also reactive. Iron is also detected extracellularly, frequently concentrated around blood vessels.
Iron accumulates abnormally in the brain regions that are typically iron-rich. In normal human brain, iron is regionally distributed and highest in the globus pallidus, substantia nigra pars reticulata, dentate nucleus, and red nucleus [25
]. In PKAN the accumulation of iron is specific to the globus pallidus and substantia nigra; a global increase in brain iron is not seen [50
]. Older neuropathological studies of tissue from NBIA patients showed that the globus pallidus and substantia nigra contain approximately three times the normal amount of iron, yet iron content is normal in other regions of the brain and in the retina and optic nerve [10
]. A more recent estimate specific to PKAN patients found a similar three-fold increase using T2 relaxometry [17
]. Systemic iron metabolism is normal as well [52
]. Iron-uptake studies in NBIA patients suggest that accumulation of iron in the basal ganglia is secondary to increased iron uptake with normal turnover [52
In regions of massive iron accumulation, spheroid bodies, many positive for iron, are also seen [30
]. Axonal spheroids are posited to represent swollen or bloated axons, possibly secondary to defects in axonal transport. They are seen in normal aging brains and in a number of other neurodegenerative disorders, including the neuroaxonal dystrophies. Other neuropathologic findings include demyelination, neuronal loss, and gliosis, which occur predominantly within the globus pallidus and substantia nigra, where focal, symmetrical destruction may be grossly evident. In addition to iron deposition and spheroid formation in the brains of people with NBIA, ceroid-lipfuscin and neuromelanin accumulate both intra and extraneuronally.
Numerous papers on NBIA report the presence of Lewy bodies and neurofibrillary tangles with accumulations of tau and alpha-synuclein [2
]. Yet, not a single case in the literature is likely to have been PKAN, based on the clinical and radiographic information available. This observation underscores the need to carefully reexamine our current knowledge of the neuropathology in NBIA in order to determine what can be applied to PKAN. Confirmed PKAN brain tissue has only recently become available and plans for further studies are underway.
Nonspecific systemic cytologic abnormalities reported in NBIA include bone marrow macrophages containing ceroid-lipofuscin and circulating lymphocytes with vacuoles and cytosomic inclusion bodies, similar to those seen in ceroid-lipofuscin storage disorders [52
]. The patients in whom these “sea-blue histiocytes” were reported likely represent the non-PKAN form of NBIA. Acanthocytes have been reported in a subset of patients with NBIA [24
], many of whom probably had PKAN. Lipofuscin and acanthocytes can both result from lipid peroxidation, a process stimulated by iron [9
]. These cytologic abnormalities, while not prominent in the pathology of this disorder, may shed light on the underlying pathophysiology of PKAN.
Since retinopathy is characteristic of PKAN, the pathology literature on this feature is likely to be specific to this disorder. Ophthalmoscopic examination in patients diagnosed with NBIA showed prominent bone spicule pigmentation and accumulation beneath the sensory retina of numerous yellowish-white globular masses [33
]. These findings were recently confirmed in a clinical study by Egan et al. of 10 patients with confirmed PKAN. Ophthalmoscopic examination showed bone spicule accumulation in four of 10 patients of varying age and disease severity. These four also had severe rod and cone function abnormalities as measured by electroretinogram (ERG). Several additional patients with normal ophthalmoscopic exams had mild or moderate ERG changes [12
]. It is unknown whether early-stage patients such as these may have sub-clinical changes detectable by pathologic examination.
On light microscopic study, there is a total loss of the outer segment of the photoreceptor cells and near total loss of the inner segment. The outer nuclear and outer plexiform layers are thinned or absent. The retinal pigment epithelium comprises a population of enlarged epithelial cells that contain both individual pigment granules and large, round pigment aggregates, a change seen primarily in the equatorial and pre-equatorial regions. Similar pigment-laden cells are present in the outer retinal layers peripheral to the perimacular area. The pigment granule clusters are melanolipofuscin complexes and represent the pathologic correlate of the yellowish-white globular masses seen on funduscopic examination [59
]. There is migration of the retinal pigment epithelium into the inner retinal layers in perivascular regions [33
], which likely accounts for the ophthalmoscopic appearance of bone spicule pigmentation. No stainable iron is seen in any part of the eye; however, focal axonal degeneration and cytoid bodies (spheroids) are noted [59
]. Abnormal accumulation of lipfuscin is reported in conjunctival fibroblasts, retinal vessel pericytes, and macrophages [33
]. Glial proliferation occurs throughout the retina and around blood vessels.
Since male mice defective for Pank2 have azoospermia [32
], we analyzed semen samples from two adult males with atypical PKAN as part of our recent clinical study. Both sperm samples were found to have similar morphologic and motility abnormalities. These included an increased frequency of tail abnormalities and amorphous sperm heads. The percentages of motile sperm with normal morphology were reduced relative to the WHO standards [65
]. Prior to knowledge of azoospermia in the mice, fertility had not been questioned or explored in the PKAN population because most patients do not reproduce due to the severity of their movement disorder and shortened lifespan.